Maize cytoplasmic male sterility (CMS) S-type restorer gene Rf3

ABSTRACT

A newly identified protein that is encoded by a polynucleotide sequence associated with cytoplasmic male sterility restorer activity (Rf3) is described. The cytoplasmic male sterility restorer gene can be inserted through breeding introgression into plant genomes to restore cytoplasmic male sterility in plants. Further applications of the newly identified polynucleotide sequence associated with cytoplasmic male sterility restorer activity include a mutation (rf3) which results in cytoplasmic male sterility. The cytoplasmic male sterility restorer gene can be inserted through breeding introgression into plant genomes to result in cytoplasmic male sterility in plants. Methods for detecting the cytoplasmic male sterility restorer (Rf3) and the cytoplasmic male sterility (rf3) gene sequences are further described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC § 119(e) of U.S.Provisional Application Ser. No. 61/922,344, filed on Dec. 31, 2013, theentire disclosure of which is incorporated herein by reference

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“74583_ST25.txt”, created on Dec. 30, 2014, and having a size of 42kilobytes and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

The disclosure relates to the isolation and identification of proteinsequences that are encoded by the cytoplasmic male sterility restorer(Rf3) and cytoplasmic male sterile (rf3) gene sequences, and for methodsof detecting the cytoplasmic male sterility restorer (Rf3) andcytoplasmic male sterile (rf3) gene sequences in plants. Furthermore,the disclosure relates to the field of plant breeding for introducingthe cytoplasmic male sterility restorer (Rf3) and cytoplasmic malesterile (rf3) gene sequences into progeny plants.

BACKGROUND OF THE INVENTION

Cytoplasmic male sterility (CMS) is the maternally inherited inabilityto produce functional pollen and has been successfully used incommercial production of hybrid seed, thus avoiding the drawbacks ofhand or mechanical emasculation. (Kaul, M. L. H. 1988. Male sterility inhigher plants. Springer-Verlag, Berlin). More than 40 sources of CMShave been identified and classified into three major groups: CMS-T(Texas), CMS-S (USDA) and CMS-C(Charrua) type cytoplasm. (Beckett, J.B., Crop Sci., 11:724-726, 1971).

The Rf restorer genes, which impart and restore fertility to plants thatare cytoplasmic male sterile, have been cloned or mapped at highresolution from several plant species. To date, eleven Rf genes havebeen cloned or mapped to high resolution. Most cloned restorer genes,except Rf2 and Rf4 in maize and Rf17 and Rf2 in rice, encode differentpentatricopeptide repeat (PPR) proteins. The PPR proteins contain two to27 repeats of 35 amino acids, called PPR motifs (Small, I. D. andPeeters, N., Trends Biochem. Sci., 25:46-47, 2000). Many PPR proteinsare targeted to mitochondria where the CMS-associated genes and productsare located (Lurin, C. et al., Plant Cell, 16:2089-2103, 2004).

In maize, the genes encoding the restorer of the S type cytoplasm of CMSbehave as a gametophytic trait. Maize plants with CMS-S type cytoplasmare restored by the single dominant gene, Rf3, which was mapped togenomic fragments of several centimorgans in length on chromosome 2, viamarker sequences. (Kamps and Chase, Theor. Appl. Genet., 95:525-531,1997; Tie, S., Xia, J., Qiu, F. and Zheng, Y. Plant Mol. Biol.,24:71-80, 2006; Zhang, Z. and Zheng Y. Mol. Gen. Genomics., 276:162-169,2006). Traditional breeding applications deploy the use of markers thatare associated at great distance with the Rf3 and rf3 alleles to restoreor to provide cytoplasmic male sterility in maize plants, respectively.(Laughnan, J. R. and Gabay, S. J. 1978. Nuclear and cytoplasmicmutations to fertility in S male-sterile maize. pp. 427-446. In: MaizeBreeding and Genetics). Furthermore, heterozygous (Rf3/rf3) CMS-S plantsare semi-fertile, shedding approximately 50% abortive collapsed pollencontaining the rf3 allele and approximately 50% starch-filled fertilepollen containing the Rf3 allele. The rf3 allele in Rf3/rf3 plantscannot be transferred to progeny through sterile pollen, and generatesterile plants in an F2 generation. (Tie et al., Plant Mol. Biol. Rep.,24: 71-80, 2006). This type of inheritance makes it difficult to collectaccurate phenotypic data from an F2 mapping population. As such,traditional methods for using or identifying the dominant and recessivealleles of the Rf3 genes are labor and time-intensive.

Therefore, there exists a need exists for compositions and methods thatcan be utilized to isolate and identify protein sequences that areencoded by the cytoplasmic male sterility restorer (Rf3) and cytoplasmicmale sterile (rf3) gene sequences, and for methods of detecting thecytoplasmic male sterility restorer (Rf3) and cytoplasmic male sterile(rf3) gene sequences in plants.

Accordingly, the present disclosure provides an alternative approach forthe isolation and identification of polynucleotide and protein sequencesencoded by the Rf3 allele that result in the cytoplasmic male sterilityrestorer phenotype, and subsequent sequence modifications encoded by therf3 allele that result in the cytoplasmic male sterile phenotype. Thediscovery of these sequences can be utilized in cytoplasmic male sterilesystems for breeding hybrid plants of a number of crop species.Furthermore, the application of this system can result in cost savingsand increased efficiency. For example, deployment of a cytoplasmic malesterile system in corn can be used to eliminate the expensive andlaborious task of detasselling corn plants to avoid self pollination.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

BRIEF SUMMARY OF THE INVENTION

The present disclosure provides, an isolated polypeptide comprising anamino acid sequence of SEQ ID NO:1. In a further embodiment the isolatedpolypeptide of SEQ ID NO:1 comprises an amino acid sequence which has atleast 85% sequence identity to SEQ ID NO:3. In aspects of an embodiment,the isolated polypeptides of SEQ ID NO:1 and SEQ ID NO:3 havecytoplasmic male sterile activity.

In other aspects, the present disclosure relates to an isolatedpolypeptide comprising an amino acid sequence of SEQ ID NO:5. In afurther embodiment, the isolated polypeptide of SEQ ID NO:5 comprises anamino acid sequence which has at least 85% sequence identity to SEQ IDNO:7. In aspects of an embodiment, the isolated polypeptides of SEQ IDNO:5 and SEQ ID NO:7 have cytoplasmic male sterile restorer activity.

In additional aspects, the subject disclosure relates to a method forproducing a progeny cytoplasmic male sterile plant. In other aspects,the subject disclosure provides methods for restoring fertility to aprogeny of a cytoplasmic male sterile plant. In a subsequent aspect, thesubject disclosure relates to a method of detecting a plant comprising acytoplasmic male sterile restorer trait. Furthermore, other aspects ofthe subject disclosure relate to a method of detecting an expressionlevel of a cytoplasmic male sterile restorer trait

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A illustrates the linkage map and QTL position of the Rf3 alleleand FIG. 1B illustrates the corresponding trait on chromosome 2.

FIG. 2 illustrates a polynucleotide sequence alignment of the Rf3 codingsequences. The sequences obtained from the 4XP811 and 7SH382 ms plantscorrespond with the cytoplasmic male sterile plants that are homozygousrecessive (rf3/rf3). The sequences obtained from the LH60 and MBB56plants correspond with the restored cytoplasmic male fertile plants thatare homozygous dominant (Rf3/Rf3). The asterisk above the alignmentindicates the base pair modification that results in the recessive rf3allele and encodes the cytoplasmic male sterile trait. This change fromSEQ ID NO:6; 5′-ATTGTTTTATTCAGT-3′ to SEQ ID NO:2; 5′-ATTGTTTTCTTCAGT-3′(wherein the underlined base pair, indicates the mutated sequence)results in the loss of restorer function and gain of cytoplasmic malesterility phenotype.

FIG. 3 illustrates a protein sequence alignment of the Rf3 codingsequences. The sequences obtained from the 4XP811 and 7SH382 ms plantscorrespond with the cytoplasmic male sterile plants that are homozygousrecessive (rf3/rf3). The sequences obtained from the LH60 and MBB56plants correspond with the restored cytoplasmic male fertile plants thatare homozygous dominant (Rf3/Rf3). The asterisk above the alignmentindicates the amino acid residue modification that results in therecessive rf3 allele and encodes the cytoplasmic male sterile trait.This change from SEQ ID NO:5; IVLFS to SEQ ID NO:1; IVFFSS (wherein theunderlined amino acid residue, indicates the mutated sequence) resultsin the loss of restorer function and gain of cytoplasmic male sterilityphenotype.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 provides the protein motif (IVFFSS) in which Leu is modifiedto a Phe (underlined), thereby resulting in the loss of restorerfunction and gain of cytoplasmic male sterility phenotype of the rf3allele.

SEQ ID NO:2 provides a polynucleotide sequence that encodes for SEQ IDNO:1.

SEQ ID NO:3 provides the protein sequence for the rf3 allele obtainedfrom the cytoplasmic male sterile corn line, Zea mays c.v. 4XP811.

SEQ ID NO:4 provides the cDNA polynucleotide sequence encoding the rf3allele obtained from the cytoplasmic male sterile corn line, Zea maysc.v. 4XP811.

SEQ ID NO:5 provides the protein motif (IVLFSS) in which conservation ofthe Leu (underlined) amino acid residue results in a restorer function,male fertile phenotype of the Rf3 allele.

SEQ ID NO:6 provides a polynucleotide sequence that encodes for SEQ IDNO:5.

SEQ ID NO:7 provides the protein sequence for the Rf3 allele obtainedfrom the restored, fertile corn line, Zea mays c.v. LH60.

SEQ ID NO:8 provides the cDNA polynucleotide sequence encoding the Rf3allele obtained from the restored, fertile corn line, Zea mays c.v.LH60.

SEQ ID NO:9 provides the protein sequence for the rf3 allele obtainedfrom the cytoplasmic male sterile corn line, Zea mays c.v. 7SH382MS.

SEQ ID NO:10 provides the cDNA polynucleotide sequence encoding the rf3allele obtained from the cytoplasmic male sterile corn line, Zea maysc.v. 7SH382MS.

SEQ ID NO:11 provides the protein sequence for the Rf3 allele obtainedfrom the restored, fertile corn line, Zea mays c.v. MBB56.

SEQ ID NO:12 provides the cDNA polynucleotide sequence encoding the Rf3allele obtained from the restored, fertile corn line, Zea mays c.v.MBB56.

SEQ ID NO:13 to SEQ ID NO:18 provide primers and probes used for thedetection of the dominant and recessive Rf3 alleles.

DETAILED DESCRIPTION I. Overview

The present disclosure provides, an isolated polypeptide comprising anamino acid sequence of SEQ ID NO:1. In a further embodiment the isolatedpolypeptide of SEQ ID NO:1 comprises an amino acid sequence which has atleast 85% sequence identity to SEQ ID NO:3. In aspects of an embodiment,the isolated polypeptides of SEQ ID NO:1 and SEQ ID NO:3 havecytoplasmic male sterile activity.

In other aspects, the present disclosure relates to an isolatedpolypeptide comprising an amino acid sequence of SEQ ID NO:5. In afurther embodiment, the isolated polypeptide of SEQ ID NO:5 comprises anamino acid sequence which has at least 85% sequence identity to SEQ IDNO:7. In aspects of an embodiment, the isolated polypeptides of SEQ IDNO:5 and SEQ ID NO:7 have cytoplasmic male sterile restorer activity.

In additional aspects, the subject disclosure relates to a method forproducing a progeny cytoplasmic male sterile plant. In other aspects,the subject disclosure provides methods for restoring fertility to aprogeny of a cytoplasmic male sterile plant. In a subsequent aspect, thesubject disclosure relates to a method of detecting a plant comprising acytoplasmic male sterile restorer trait. Furthermore, other aspects ofthe subject disclosure relate to a method of detecting an expressionlevel of a cytoplasmic male sterile restorer trait.

II. Clause List

Provided herein are embodiment of the invention, further described bythe following enumerated clauses:

1. An isolated polypeptide comprising an amino acid sequence of SEQ IDNO: 1.

2. An isolated polypeptide consisting of an amino acid sequence of SEQID NO: 1.

3. An isolated polypeptide consisting essentially of an amino acidsequence of SEQ ID NO: 1.

4. The isolated polypeptide of clause 1 comprising an amino acidsequence which has at least 85% sequence identity to SEQ ID NO: 3.

5. The isolated polypeptide of clause 1 consisting of an amino acidsequence which has at least 85% sequence identity to SEQ ID NO: 3.

6. The isolated polypeptide of clause 1 consisting essentially of anamino acid sequence which has at least 85% sequence identity to SEQ IDNO: 3.

7. The isolated polypeptide sequence of clause 1 or clause 4 wherein thepolypeptide has cytoplasmic male sterile activity.

8. A cell comprising the polypeptide of clause 1.

9. The cell of clause 8, wherein the cell is a plant cell.

10. A plant comprising the plant cell of clause 9.

11. The plant of clause 10, wherein the plant is a monocotyledonousplant.

12. The plant of clause 11, wherein the monocotyledonous plant is amaize plant.

13. A seed obtained from the plant of clause 10.

14. A synthetic nucleic acid sequence encoding the polypeptide of clause1.

15. The synthetic nucleic acid sequence of clause 14 comprising thesequence of SEQ ID NO:2.

16. The synthetic nucleic acid sequence of clause 14 consisting of thesequence of SEQ ID NO:2.

17. The synthetic nucleic acid sequence of clause 14 consistingessentially of the sequence of SEQ ID NO:2.

18. The synthetic nucleic acid sequence of clause 14 comprising at least90% sequence identity to the sequence of SEQ ID NO:4.

19. The synthetic nucleic acid sequence of clause 14 consisting of thesequence of SEQ ID NO:4.

20. The synthetic nucleic acid sequence of clause 14 consistingessentially of the sequence of SEQ ID NO:4.

21. A polynucleotide comprising the synthetic nucleic acid sequence ofclause 14.

22. The polynucleotide of clause 21, wherein the polynucleotide isoperably linked to a promoter, wherein the promoter is functional inplants.

23. The polynucleotide of clause 22, wherein the promoter is a plantcytoplasmic male sterile promoter.

24. A cell comprising the polynucleotide of clause 21.

25. A vector comprising the synthetic nucleic acid sequence of clause14.

26. A cell comprising the vector of clause 25.

27. The isolated polypeptide of clause 1 comprising an amino acidsequence which has at least 85% sequence identity to SEQ ID NO: 9.

28. The isolated polypeptide of clause 1 consisting of an amino acidsequence which has at least 85% sequence identity to SEQ ID NO: 9.

29. The isolated polypeptide of clause 1 consisting essentially of anamino acid sequence which has at least 85% sequence identity to SEQ IDNO: 9.

30. The isolated polypeptide sequence of clause 27, wherein thepolypeptide has cytoplasmic male sterile activity.

31. A cell comprising the polypeptide of clause 27.

32. The cell of clause 31, wherein the cell is a plant cell.

33. A plant comprising the plant cell of clause 32.

34. The plant of clause 33, wherein the plant is a monocotyledonousplant.

35. The plant of clause 34, wherein the monocotyledonous plant is amaize plant.

36. A seed obtained from the plant of clause 33.

37. A synthetic nucleic acid sequence encoding the polypeptide of clause27.

38. The synthetic nucleic acid sequence of clause 37 comprising at least90% sequence identity to the sequence of SEQ ID NO:10.

39. The synthetic nucleic acid sequence of clause 37 consisting of thesequence of SEQ ID NO:10.

40. The synthetic nucleic acid sequence of clause 37 consistingessentially of the sequence of SEQ ID NO:10.

41. A polynucleotide comprising the synthetic nucleic acid sequence ofclause 37.

42. The polynucleotide of clause 41, wherein the polynucleotide isoperably linked to a promoter, wherein the promoter is functional inplants.

43. The polynucleotide of clause 42, wherein the promoter is a plantcytoplasmic male sterile promoter.

44. A cell comprising the polynucleotide of clause 41.

45. A vector comprising the synthetic nucleic acid sequence of clause37.

46. A cell comprising the vector of clause 45.

47. The plant of clause 35, wherein the plant has CMS-S type cytoplasm.

48. A method for producing a progeny cytoplasmic male sterile plant, themethod comprising the steps of:

-   -   a) crossing a female parent plant with a male parent plant,        wherein the female parent plant is a cytoplasmic male sterile        parent plant homozygous for SEQ ID NO:4, and wherein the male        parent plant is a fertile parent plant;    -   b) harvesting a progeny seed from the cross of step (a), wherein        the progeny seed is homozygous for SEQ ID NO:4;    -   c) planting the progeny seed; and,    -   d) growing the progeny seed, wherein the progeny seed produce        the progeny cytoplasmic male sterile plant, wherein the progeny        cytoplasmic male sterile plant is homozygous for SEQ ID NO:4.

49. The method for producing a progeny cytoplasmic male sterile plant ofclause 48, wherein the female and male parent plants aremonocotyledonous plants.

50. The method for producing a progeny cytoplasmic male sterile plant ofclause 49, wherein the monocotyledonous plants are maize plants.

51. The method for producing a progeny cytoplasmic male sterile plant ofclause 48, wherein the male parent plant is isogenic to the femaleparent plant.

52. The method for producing a progeny cytoplasmic male sterile plant ofclause 48, wherein the male parent plant is homozygous or heterozygousfor SEQ ID NO:8.

53. The method for producing a progeny cytoplasmic male sterile plant ofclause 48, the method further comprising introducing a desired traitinto the progeny cytoplasmic male sterile plant.

54. The method for producing a progeny cytoplasmic male sterile plant ofclause 48, the method further comprising the steps of:

-   -   e) crossing the progeny cytoplasmic male sterile plant, with        another plant comprising a desired trait to produce F1 progeny        plants;    -   f) selecting F1 progeny plants that have the desired trait to        produce selected F1 progeny plants;    -   g) crossing the selected F1 progeny plants with the progeny        cytoplasmic male sterile plant to produce backcross progeny        plants;    -   h) selecting for backcross progeny plants that have the desired        trait; and,    -   i) repeating steps (g) and (h) three or more times in succession        to produce selected fourth or higher backcross progeny plants        that comprise the desired trait.

55. The method for producing a progeny cytoplasmic male sterile plant of53, wherein the desired trait is selected from the group consisting ofan insecticidal resistance trait, herbicide tolerant trait, diseaseresistance trait, yield increase trait, nutritional quality trait,agronomic increase trait, and combinations thereof.

56. A method for producing a progeny cytoplasmic male sterile plant, themethod comprising the steps of:

-   -   a) crossing a female parent plant with a male parent plant,        wherein the female parent plant is a cytoplasmic male sterile        parent plant homozygous for SEQ ID NO:10, and wherein the male        parent plant is a fertile parent plant;    -   b) harvesting a progeny seed from the cross of step (a), wherein        the progeny seed is homozygous for SEQ ID NO:10;    -   c) planting the progeny seed; and,    -   d) growing the progeny seed, wherein the progeny seed produce        the progeny cytoplasmic male sterile plant, wherein the progeny        cytoplasmic male sterile plant is homozygous for SEQ ID NO:10.

57. The method for producing a progeny cytoplasmic male sterile plant ofclause 56, wherein the female and male parent plants aremonocotyledonous plants.

58. The method for producing a progeny cytoplasmic male sterile plant ofclause 57, wherein the monocotyledonous plants are maize plants.

59. The method for producing a progeny cytoplasmic male sterile plant ofclause 56, wherein the male parent plant is isogenic to the femaleparent plant.

60. The method for producing a progeny cytoplasmic male sterile plant ofclause 56, wherein the male parent plant is homozygous or heterozygousfor SEQ ID NO:8.

61. The method for producing a progeny cytoplasmic male sterile plant ofclause 56, the method further comprising introducing a desired traitinto the progeny cytoplasmic male sterile plant.

62. The method for producing a progeny cytoplasmic male sterile plant ofclause 56, the method further comprising the steps of:

-   -   e) crossing the progeny cytoplasmic male sterile plant, with        another plant comprising a desired trait to produce F1 progeny        plants;    -   f) selecting F1 progeny plants that have the desired trait to        produce selected F1 progeny plants;    -   g) crossing the selected F1 progeny plants with the progeny        cytoplasmic male sterile plant to produce backcross progeny        plants;    -   h) selecting for backcross progeny plants that have the desired        trait; and,    -   i) repeating steps (g) and (h) three or more times in succession        to produce selected fourth or higher backcross progeny plants        that comprise the desired trait.

63. The method for producing a progeny cytoplasmic male sterile plant of61, wherein the desired trait is selected from the group consisting ofan insecticidal resistance trait, herbicide tolerant trait, diseaseresistance trait, yield increase trait, nutritional quality trait,agronomic increase trait, and combinations thereof.

64. A method for restoring fertility to a progeny of a cytoplasmic malesterile parent plant, the method comprising:

-   -   a) crossing a female parent plant with a male parent plant,        wherein the female parent plant is a cytoplasmic male sterile        parent plant homozygous or heterozygous for SEQ ID NO:8, and        wherein the cytoplasmic male sterile plant is a fertile parent        plant;    -   b) harvesting a progeny seed from the cross of step (a), wherein        the progeny seed is homozygous or heterozygous for SEQ ID NO:8;    -   c) planting the progeny seed;    -   d) growing the progeny seed, wherein the progeny seed produce a        progeny cytoplasmic male fertile plant; and,    -   e) restoring fertility to the progeny of the cytoplasmic male        sterile parent plant, wherein the progeny cytoplasmic male        fertile plant is homozygous or heterozygous for SEQ ID NO:8.

65. The method for restoring fertility to a progeny of a cytoplasmicmale sterile parent plant of clause 64, wherein the female and maleparent plants are monocotyledonous plants.

66. The method for restoring fertility to a progeny of a cytoplasmicmale sterile parent plant of clause 65, wherein the monocotyledonousplants are maize plants.

67. The method for restoring fertility to a progeny of a cytoplasmicmale sterile parent plant of clause 64, wherein the male parent plant isisogenic to the female parent plant.

68. The method for restoring fertility to a progeny of a cytoplasmicmale sterile parent plant of clause 64, wherein the male parent plant ishomozygous or heterozygous for SEQ ID NO:8.

69. The method for restoring fertility to a progeny of a cytoplasmicmale sterile parent plant of clause 64, the method further comprisingintroducing a desired trait into the cytoplasmic male sterile plant.

70. The method for restoring fertility to a progeny of a cytoplasmicmale sterile parent plant of clause 64, the method further comprising:

-   -   f) crossing the progeny cytoplasmic male sterile plant, with        another plant comprising a desired trait to produce F1 progeny        plants;    -   g) selecting F1 progeny plants that have the desired trait to        produce selected F1 progeny plants;    -   h) crossing the selected F1 progeny plants with progeny        cytoplasmic male sterile plant to produce backcross progeny        plants;    -   i) selecting for backcross progeny plants that have the desired        trait; and,    -   j) repeating steps (h) and (i) three or more times in succession        to produce selected fourth or higher backcross progeny plants        that comprise the desired trait.

71. The method for restoring fertility to a progeny of a cytoplasmicmale sterile parent plant of clause 69, wherein the desired trait isselected from the group consisting of an insecticidal resistance trait,herbicide tolerant trait, disease resistance trait, yield increasetrait, nutritional quality trait, agronomic increase trait, andcombinations thereof.

72. An isolated polypeptide comprising an amino acid sequence of SEQ IDNO: 5.

73. An isolated polypeptide consisting of an amino acid sequence of SEQID NO: 5.

74. An isolated polypeptide consisting essentially of an amino acidsequence of SEQ ID NO: 5.

75. The isolated polypeptide of clause 72 comprising an amino acidsequence which has at least 85% sequence identity to SEQ ID NO: 7.

76. The isolated polypeptide of clause 72 consisting of an amino acidsequence which has at least 85% sequence identity to SEQ ID NO: 7.

77. The isolated polypeptide of clause 72 consisting essentially of anamino acid sequence which has at least 85% sequence identity to SEQ IDNO: 7.

78. The isolated polypeptide sequence of clause 72 or clause 75, whereinthe polypeptide has cytoplasmic male sterile restorer activity.

79. A cell comprising the polypeptide of clause 72.

80. The cell of clause 79, wherein the cell is a plant cell.

81. A plant comprising the plant cell of clause 80.

82. The plant of clause 81, wherein the plant is a monocotyledonousplant.

83. The plant of clause 82, wherein the monocotyledonous plant is amaize plant.

84. The plant of clause 83, wherein the plant has CMS-S type cytoplasm.

85. A seed obtained from the plant of clause 81.

86. A synthetic nucleic acid sequence encoding the polypeptide of clause72.

87. The synthetic nucleic acid sequence of clause 86 comprising thesequence of SEQ ID NO:8.

88. The synthetic nucleic acid sequence of clause 86 consisting of thesequence of SEQ ID NO:8.

89. The synthetic nucleic acid sequence of clause 86 consistingessentially of the sequence of SEQ ID NO:8.

90. The synthetic nucleic acid sequence of clause 86 comprising thesequence of SEQ ID NO:6.

91. The synthetic nucleic acid sequence of clause 86 consisting of thesequence of SEQ ID NO:6.

92. The synthetic nucleic acid sequence of clause 86 consistingessentially of the sequence of SEQ ID NO:6.

93. A gene expression cassette comprising the synthetic nucleic acidsequence of clause 86.

94. The gene expression cassette of clause 93, wherein the syntheticnucleic acid sequence is operably linked to a promoter, wherein thepromoter is functional in plants.

95. The gene expression cassette of clause 94, wherein the promoter is aplant cytoplasmic male sterile promoter.

96. A cell comprising the gene expression cassette of clause 93.

97. A vector comprising the synthetic nucleic acid sequence of clause86.

98. A cell comprising the vector of clause 97.

99. A method for restoring fertility to a cytoplasmic male sterileplant, the method comprising the steps of;

-   -   a) transforming the cytoplasmic male sterile plant with the        synthetic nucleic acid sequence of clause 86;    -   b) integrating the synthetic nucleic acid sequence into the        genome of the cytoplasmic male sterile plant; and,    -   c) expressing the synthetic nucleic acid sequence, wherein        expression of the synthetic nucleic acid sequence restores        fertility to the cytoplasmic male sterile plant.

100. A method for altering plant morphology in a cytoplasmic malesterile plant, the method comprising the steps of;

-   -   a) transforming the cytoplasmic male sterile plant with the        synthetic nucleic acid sequence of clause 86;    -   b) integrating the synthetic nucleic acid sequence into the        genome of the cytoplasmic male sterile plant; and,    -   c) expressing the synthetic nucleic acid sequence, wherein        expression of the synthetic nucleic acid sequence alters plant        morphology in the cytoplasmic male sterile plant.

101. A method of detecting a plant comprising a cytoplasmic male sterilerestorer trait, the method comprising the steps of:

-   -   a) isolating a genomic polynucleotide sample from a plant, plant        tissue, plant part, or plant cell;    -   b) adding a set of oligonucleotide primers to the genomic        polynucleotide sample;    -   c) subjecting the genomic polynucleotide sample and the set of        oligonucleotide primers to an amplification process; and,    -   d) detecting at least one amplified product, wherein the        amplified product indicates the presence of the cytoplasmic male        sterile restorer trait in the plant.

102. The method of detecting a plant comprising a cytoplasmic malesterile restorer trait of clause 101, wherein the set of oligonucleotideprimers are selected from the group consisting of SEQ ID NO:28, SEQ IDNO:29, and SEQ ID NO:30.

103. The method of detecting a plant comprising a cytoplasmic malesterile restorer trait of clause 101, wherein the amplified productcomprises SEQ ID NO:6.

104. The method of detecting a plant comprising a cytoplasmic malesterile restorer trait of clause 101, wherein the amplified productconsists of SEQ ID NO:6.

105. The method of detecting a plant comprising a cytoplasmic malesterile restorer trait of clause 101, wherein the amplified productconsists essentially of SEQ ID NO:6.

106. The method of detecting a plant comprising a cytoplasmic malesterile restorer trait of clause 101, wherein the amplified productcomprises a fragment of SEQ ID NO:8.

107. The method of detecting a plant comprising a cytoplasmic malesterile restorer trait of clause 101, wherein the amplified productconsists of a fragment of SEQ ID NO:8.

108. The method of detecting a plant comprising a cytoplasmic malesterile restorer trait of clause 101, wherein the amplified productconsists essentially of a fragment of SEQ ID NO:8.

109. The method of detecting a plant comprising a cytoplasmic malesterile restorer trait of clause 101, wherein the amplified productcomprises a fragment of SEQ ID NO:12.

110. The method of detecting a plant comprising a cytoplasmic malesterile restorer trait of clause 101, wherein the amplified productconsists of a fragment of SEQ ID NO:12.

111. The method of detecting a plant comprising a cytoplasmic malesterile restorer trait of clause 101, wherein the amplified productconsists essentially of a fragment of SEQ ID NO:12.

112. The method of detecting a plant comprising a cytoplasmic malesterile restorer trait of clause 101, wherein the amplified product isquantitated.

113. The method of detecting an expression level of a cytoplasmic malesterile restorer trait, the method comprising the steps of:

-   -   a) performing a first amplification process using a cytoplasmic        male sterile restorer trait probe (SEQ ID NO:30), a forward        cytoplasmic male sterile restorer trait primer (SEQ ID NO:28)        and a reverse cytoplasmic male sterile restorer trait primer        (SEQ ID NO:29) on a genomic polynucleotide sample, wherein a        first fluorescent dye is released from the cytoplasmic male        sterile restorer trait probe (SEQ ID NO:30);    -   b) performing a second amplification process using a reference        probe (SEQ ID NO:36), a forward reference primer (SEQ ID NO:34)        and a reverse reference primer (SEQ ID NO:35) on the genomic        polynucleotide sample, wherein a second fluorescent dye is        released from the reference probe (SEQ ID NO:36); and,    -   c) quantitating the relative intensity of the first fluorescent        dye to the second fluorescent dye to detect the expression level        of the cytoplasmic male sterile restorer trait.

114. The method of detecting an expression level of a cytoplasmic malesterile restorer trait of clause 113, wherein the first and secondamplification processes are performed in a single PCR assay tube usingprobes with different fluorescent dyes.

115. The method of detecting an expression level of a cytoplasmic malesterile restorer trait of clause 113, the method further comprising thesteps of:

-   -   d) loading a PCR solution in a single PCR assay tube, the PCR        solution comprising a polymerase with 5′ to 3′ nuclease        activity, deoxynucleotides, the primers, the probes, a buffer,        and the genomic polynucleotide sample;    -   e) amplifying the PCR solution, wherein the PCR solution is        treated under amplification conditions such that the 5′ to 3′        nuclease activity of the polymerase cleaves the probes thereby        releasing the fluorescent dye that emits light; and,    -   f) measuring the light emitted from fluorescent dye, during the        amplification.

116. The method of detecting an expression level of a cytoplasmic malesterile restorer trait of clause 113, wherein the cytoplasmic malesterile trait probe comprises the first fluorescent dye and a firstquencher.

117. The method of detecting an expression level of a cytoplasmic malesterile restorer trait of clause 116, wherein the first fluorescent dyeis selected from the group consisting of a HEX fluorescent dye, a VICfluorescent dye, a FAM fluorescent dye, a JOE fluorescent dye, a TETfluorescent dye, a Cy 3 fluorescent dye, a Cy 3.5 fluorescent dye, a Cy5 fluorescent dye, a Cy 5.5 fluorescent dye, a Cy 7 fluorescent dye, ora ROX fluorescent dye.

118. The method of detecting an expression level of a cytoplasmic malesterile restorer trait of clause 116, wherein the first quencher isselected from the group consisting of a Dabcyl quencher, a Tamraquencher, a Qxl quencher, an Iowa Black FQ quencher, an Iowa Black RQquencher, an IR Dye QC-1 quencher, a MGB quencher, or a Blackholequencher.

119. The method of detecting an expression level of a cytoplasmic malesterile restorer trait of clause 113, wherein the reference probecomprises the second fluorescent dye and a second quencher.

120. The method of detecting an expression level of a cytoplasmic malesterile restorer trait of clause 119, wherein the second fluorescent dyeis selected from the group consisting of a HEX fluorescent dye, a FAMfluorescent dye, a VIC fluorescent dye, a JOE fluorescent dye, a TETfluorescent dye, a Cy 3 fluorescent dye, a Cy 3.5 fluorescent dye, a Cy5 fluorescent dye, a Cy 5.5 fluorescent dye, a Cy 7 fluorescent dye, ora ROX fluorescent dye.

121. The method of detecting an expression level of a cytoplasmic malesterile restorer trait of clause 119, wherein the second quencher isselected from the group consisting of a Dabcyl quencher, a Tamraquencher, a Qxl quencher, an Iowa Black FQ quencher, an Iowa Black RQquencher, an IR Dye QC-1 quencher, a MGB quencher, or a Blackholequencher.

122. The method of detecting an expression level of a cytoplasmic malesterile restorer trait of clause 113 or clause 101, wherein the genomicpolynucleotide sample comprises cDNA produced from mRNA isolated from aplant, plant tissue, plant part, or plant cell.

123. A cell comprising the polypeptide of clause 4.

124. The cell of clause 123, wherein the cell is a plant cell.

125. A plant comprising the plant cell of clause 124.

126. The plant of clause 125, wherein the plant is a monocotyledonousplant.

127. The plant of clause 126, wherein the monocotyledonous plant is amaize plant.

128. A seed obtained from the plant of clause 125.

129. A synthetic nucleic acid sequence encoding the polypeptide ofclause 4.

130. The synthetic nucleic acid sequence of clause 129 comprising asequence with at least 90% sequence identity to the sequence of SEQ IDNO:4.

131. The synthetic nucleic acid sequence of clause 129 consisting of thesequence of SEQ ID NO:4.

132. The synthetic nucleic acid sequence of clause 129 consistingessentially of the sequence of SEQ ID NO:4.

133. A polynucleotide comprising the synthetic nucleic acid sequence ofclause 129.

134. The polynucleotide of clause 133, wherein the polynucleotide isoperably linked to a promoter, wherein the promoter is functional inplants.

135. The polynucleotide of clause 134, wherein the promoter is a plantcytoplasmic male sterile promoter.

136. A cell comprising the polynucleotide of clause 133.

137. A vector comprising the synthetic nucleic acid sequence of clause129.

138. A cell comprising the vector of clause 137.

139. The plant of clause 125, wherein the plant has CMS-S typecytoplasm.

III. Terms

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure relates. In case of conflict, thepresent application including the definitions will control. Unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. All publications, patentsand other references mentioned herein are incorporated by reference intheir entireties for all purposes as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference, unless only specific sections of patents orpatent publications are indicated to be incorporated by reference.

In order to further clarify this disclosure, the following terms,abbreviations and definitions are provided.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains,” or “containing,” or any othervariation thereof, are intended to be non-exclusive or open-ended. Forexample, a composition, a mixture, a process, a method, an article, oran apparatus that comprises a list of elements is not necessarilylimited to only those elements but may include other elements notexpressly listed or inherent to such composition, mixture, process,method, article, or apparatus. Further, unless expressly stated to thecontrary, “or” refers to an inclusive or and not to an exclusive or. Forexample, a condition A or B is satisfied by any one of the following: Ais true (or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the indefinite articles “a” and “an” preceding an element orcomponent of an embodiment of the disclosure are intended to benonrestrictive regarding the number of instances, i.e., occurrences ofthe element or component. Therefore “a” or “an” should be read toinclude one or at least one, and the singular word form of the elementor component also includes the plural unless the number is obviouslymeant to be singular.

The term “invention” or “present invention” as used herein is anon-limiting term and is not intended to refer to any single embodimentof the particular invention but encompasses all possible embodiments asdisclosed in the application.

As used herein, the term “plant” includes a whole plant and anydescendant, cell, tissue, or part of a plant. The term “plant parts”include any part(s) of a plant, including, for example and withoutlimitation: seed (including mature seed and immature seed); a plantcutting; a plant cell; a plant cell culture; a plant organ (e.g.,pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, andexplants). A plant tissue or plant organ may be a seed, protoplast,callus, or any other group of plant cells that is organized into astructural or functional unit. A plant cell or tissue culture may becapable of regenerating a plant having the physiological andmorphological characteristics of the plant from which the cell or tissuewas obtained, and of regenerating a plant having substantially the samegenotype as the plant. In contrast, some plant cells are not capable ofbeing regenerated to produce plants. Regenerable cells in a plant cellor tissue culture may be embryos, protoplasts, meristematic cells,callus, pollen, leaves, anthers, roots, root tips, silk, flowers,kernels, ears, cobs, husks, or stalks.

Plant parts include harvestable parts and parts useful for propagationof progeny plants. Plant parts useful for propagation include, forexample and without limitation: seed; fruit; a cutting; a seedling; atuber; and a rootstock. A harvestable part of a plant may be any usefulpart of a plant, including, for example and without limitation: flower;pollen; seedling; tuber; leaf; stem; fruit; seed; and root.

A plant cell is the structural and physiological unit of the plant, andincludes protoplast cells without a cell wall and plant cells with acell wall. A plant cell may be in the form of an isolated single cell,or an aggregate of cells (e.g., a friable callus and a cultured cell),and may be part of a higher organized unit (e.g., a plant tissue, plantorgan, and plant). Thus, a plant cell may be a protoplast, a gameteproducing cell, or a cell or collection of cells that can regenerateinto a whole plant. As such, a seed, which comprises multiple plantcells and is capable of regenerating into a whole plant, is considered a“plant cell” in embodiments herein.

As used herein, the term “isolated” refers to a biological component(including a nucleic acid or protein) that has been separated, producedapart from other biological components in the cell of the organism inwhich the component naturally occurs (i.e., other chromosomal andextra-chromosomal DNA and RNA, and proteins).

As used herein, the term “purified” in reference to nucleic acidmolecules does not require absolute purity (such as a homogeneouspreparation); instead, it represents an indication that the sequence isrelatively more pure than in its native cellular environment (comparedto the natural level this level should be at least 2-5 fold greater,e.g., in terms of concentration or gene expression levels). The claimedDNA molecules obtained directly from total DNA or from total RNA. Inaddition, cDNA clones are not naturally occurring, but rather arepreferably obtained via manipulation of a partially purified, naturallyoccurring substance (messenger RNA). The construction of a cDNA libraryfrom mRNA involves the creation of a library. Individual cDNA clones canbe produced from the library by clonal selection of the cells carryingthe cDNA library. Thus, the process which includes the construction of acDNA library from mRNA and selection of distinct cDNA clones yields anapproximately 10⁶-fold purification of the native message. Likewise, apromoter or gene DNA sequence could be cloned into a plasmid. Such aclone is not naturally occurring, but rather is preferably obtained viamanipulation of a partially purified, naturally occurring substance suchas a genomic DNA library. Thus, purification of at least one order ofmagnitude, preferably two or three orders, and more preferably four orfive orders of magnitude is favored in these techniques.

Similarly, synthetic represents an indication that a chemical orfunctional change in the component DNA sequence has occurred. Nucleicacid molecules and proteins that have been “synthesized” include nucleicacid molecules and proteins generated by PCR amplification or byrecombinant methods, wherein an isolated polynucleotide is furthermodified by the incorporation within a plasmid or vector. The term“synthetic” also embraces nucleic acids and proteins prepared byrecombinant DNA methods in a host cell (e.g., plant cells), as well aschemically-synthesized nucleic acid molecules, proteins, and peptides.

In engineering a gene for expression in plants, the codon bias of theprospective host plant(s) may be determined, for example, through use ofpublicly available DNA sequence databases to find information about thecodon distribution of plant genomes or the protein coding regions ofvarious plant genes. Once an optimized (e.g., a plant-optimized) DNAsequence has been designed on paper, or in silico, actual DNA moleculesmay be synthesized in the laboratory to correspond in sequence preciselyto the designed sequence. Such synthetic nucleic acid molecule moleculescan be cloned and otherwise manipulated exactly as if they were derivedfrom natural or native sources.

As used herein, the terms “polynucleotide,” “nucleic acid,” and “nucleicacid molecule” are used interchangeably, and may encompass a singularnucleic acid; plural nucleic acids; a nucleic acid fragment, variant, orderivative thereof; and nucleic acid construct (e.g., messenger RNA(mRNA) and plasmid DNA (pDNA)). A polynucleotide or nucleic acid maycontain the nucleotide sequence of a full-length cDNA sequence, or afragment thereof, including untranslated 5′ and/or 3′ sequences andcoding sequence(s). A polynucleotide or nucleic acid may be comprised ofany polyribonucleotide or polydeoxyribonucleotide, which may includeunmodified ribonucleotides or deoxyribonucleotides or modifiedribonucleotides or deoxyribonucleotides. For example, a polynucleotideor nucleic acid may be comprised of single- and double-stranded DNA; DNAthat is a mixture of single- and double-stranded regions; single- anddouble-stranded RNA; and RNA that is mixture of single- anddouble-stranded regions. Hybrid molecules comprising DNA and RNA may besingle-stranded, double-stranded, or a mixture of single- anddouble-stranded regions. The foregoing terms also include chemically,enzymatically, and metabolically modified forms of a polynucleotide ornucleic acid.

It is understood that a specific DNA refers also to the complementthereof, the sequence of which is determined according to the rules ofdeoxyribonucleotide base-pairing.

As used herein, the term “gene” refers to a nucleic acid that encodes afunctional product (RNA or polypeptide/protein). A gene may includeregulatory sequences preceding (5′ non-coding sequences) and/orfollowing (3′ non-coding sequences) the sequence encoding the functionalproduct.

As used herein, the term “coding sequence” refers to a nucleic acidsequence that encodes a specific amino acid sequence. A “regulatorysequence” refers to a nucleotide sequence located upstream (e.g., 5′non-coding sequences), within, or downstream (e.g., 3′ non-codingsequences) of a coding sequence, which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Regulatory sequences include, for example and withoutlimitation: promoters; translation leader sequences; introns;polyadenylation recognition sequences; RNA processing sites; effectorbinding sites; and stem-loop structures.

As used herein, the term “polypeptide” includes a singular polypeptide,plural polypeptides, and fragments thereof. This term refers to amolecule comprised of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length or size of the product. Accordingly, peptides,dipeptides, tripeptides, oligopeptides, protein, amino acid chain, andany other term used to refer to a chain or chains of two or more aminoacids, are included within the definition of “polypeptide,” and theforegoing terms are used interchangeably with “polypeptide” herein. Apolypeptide may be isolated from a natural biological source or producedby recombinant technology, but a specific polypeptide is not necessarilytranslated from a specific nucleic acid. A polypeptide may be generatedin any appropriate manner, including for example and without limitation,by chemical synthesis.

As used herein, the term “native” refers to the form of apolynucleotide, gene or polypeptide that is found in nature with its ownregulatory sequences, if present. The term “endogenous” refers to thenative form of the polynucleotide, gene or polypeptide in its naturallocation in the organism or in the genome of the organism.

In contrast, the term “heterologous” refers to a polynucleotide, gene orpolypeptide that is not normally found at its location in the reference(host) organism. For example, a heterologous nucleic acid may be anucleic acid that is normally found in the reference organism at adifferent genomic location. By way of further example, a heterologousnucleic acid may be a nucleic acid that is not normally found in thereference organism. A host organism comprising a hetereologouspolynucleotide, gene or polypeptide may be produced by introducing theheterologous polynucleotide, gene or polypeptide into the host organism.In particular examples, a heterologous polynucleotide comprises a nativecoding sequence, or portion thereof, that is reintroduced into a sourceorganism in a form that is different from the corresponding nativepolynucleotide. In particular examples, a heterologous gene comprises anative coding sequence, or portion thereof, that is reintroduced into asource organism in a form that is different from the correspondingnative gene. For example, a heterologous gene may include a nativecoding sequence that is a portion of a chimeric gene includingnon-native regulatory regions that is reintroduced into the native host.In particular examples, a heterologous polypeptide is a nativepolypeptide that is reintroduced into a source organism in a form thatis different from the corresponding native polypeptide.

A heterologous gene or polypeptide may be a gene or polypeptide thatcomprises a functional polypeptide or nucleic acid sequence encoding afunctional polypeptide that is fused to another genes or polypeptide toproduce a chimeric or fusion polypeptide, or a gene encoding the same.Genes and proteins of particular embodiments include specificallyexemplified full-length sequences and portions, segments, fragments(including contiguous fragments and internal and/or terminal deletionscompared to the full-length molecules), variants, mutants, chimerics,and fusions of these sequences.

As used herein, the term “modification” may refer to a change in aparticular reference polynucleotide that results in reduced,substantially eliminated, or eliminated activity of a polypeptideencoded by the reference polynucleotide. A modification may also referto a change in a reference polypeptide that results in reduced,substantially eliminated, or eliminated activity of the referencepolypeptide. Alternatively, the term “modification” may refer to achange in a reference polynucleotide that results in increased orenhanced activity of a polypeptide encoded by the referencepolynucleotide, as well as a change in a reference polypeptide thatresults in increased or enhanced activity of the reference polypeptide.Changes such as the foregoing may be made by any of several methodswell-known in the art including, for example and without limitation:deleting a portion of the reference molecule; mutating the referencemolecule (e.g., via spontaneous mutagenesis, via random mutagenesis, viamutagenesis caused by mutator genes, and via transposon mutagenesis);substituting a portion of the reference molecule; inserting an elementinto the reference molecule; down-regulating expression of the referencemolecule; altering the cellular location of the reference molecule;altering the state of the reference molecule (e.g., via methylation of areference polynucleotide, and via phosphorylation or ubiquitination of areference polypeptide); removing a cofactor of the reference molecule;introduction of an antisense RNA/DNA targeting the reference molecule;introduction of an interfering RNA/DNA targeting the reference molecule;chemical modification of the reference molecule; covalent modificationof the reference molecule; irradiation of the reference molecule with UVradiation or X-rays; homologous recombination that alters the referencemolecule; mitotic recombination that alters the reference molecule;replacement of the promoter of the reference molecule; and/orcombinations of any of the foregoing.

Guidance in determining which nucleotides or amino acid residues may bemodified in a specific example may be found by comparing the sequence ofthe reference polynucleotide or polypeptide with that of homologous(e.g., homologous yeast or bacterial) polynucleotides or polypeptides,and maximizing the number of modifications made in regions of highhomology (conserved regions) or consensus sequences.

The term “promoter” refers to a DNA sequence capable of controlling theexpression of a nucleic acid coding sequence or functional RNA. Inexamples, the controlled coding sequence is located 3′ to a promotersequence. A promoter may be derived in its entirety from a native gene,a promoter may be comprised of different elements derived from differentpromoters found in nature, or a promoter may even comprise synthetic DNAsegments. It is understood by those skilled in the art that differentpromoters can direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental or physiological conditions. Examples of all ofthe foregoing promoters are known and used in the art to control theexpression of heterologous nucleic acids. Promoters that direct theexpression of a gene in most cell types at most times are commonlyreferred to as “constitutive promoters.” Furthermore, while those in theart have (in many cases unsuccessfully) attempted to delineate the exactboundaries of regulatory sequences, it has come to be understood thatDNA fragments of different lengths may have identical promoter activity.The promoter activity of a particular nucleic acid may be assayed usingtechniques familiar to those in the art.

The term “operably linked” refers to an association of nucleic acidsequences on a single nucleic acid, wherein the function of one of thenucleic acid sequences is affected by another. For example, a promoteris operably linked with a coding sequence when the promoter is capableof effecting the expression of that coding sequence (e.g., the codingsequence is under the transcriptional control of the promoter). A codingsequence may be operably linked to a regulatory sequence in a sense orantisense orientation.

The term “expression” or “expressing,” as used herein, may refer to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from a DNA. Expression may also refer to translation of mRNAinto a polypeptide. As used herein, the term “overexpression” refers toexpression that is higher than endogenous expression of the same gene ora related gene. Thus, a heterologous gene is “overexpressed” if itsexpression is higher than that of a comparable endogenous gene.

As used herein, the term “transformation” or “transforming” refers tothe transfer and integration of a nucleic acid or fragment thereof intoa host organism, resulting in genetically stable inheritance. Hostorganisms containing a transforming nucleic acid are referred to as“transgenic,” “recombinant,” or “transformed” organisms. Known methodsof transformation include, for example: Agrobacterium tumefaciens- or A.rhizogenes-mediated transformation; calcium phosphate transformation;polybrene transformation; protoplast fusion; electroporation; ultrasonicmethods (e.g., sonoporation); liposome transformation; microinjection;transformation with naked DNA; transformation with plasmid vectors;transformation with viral vectors; biolistic transformation(microparticle bombardment); silicon carbide WHISKERS-mediatedtransformation; aerosol beaming; and PEG-mediated transformation.

As used herein, the term “introduced” (in the context of introducing anucleic acid into a cell) includes transformation of a cell, as well ascrossing a plant comprising the nucleic acid with a second plant, suchthat the second plant contains the nucleic acid, as may be performedutilizing conventional plant breeding techniques. Such breedingtechniques are known in the art. For a discussion of plant breedingtechniques, see Poehlman (1995) Breeding Field Crops, 4th Edition, AVIPublication Co., Westport Conn.

Backcrossing methods may be used to introduce a nucleic acid into aplant. This technique has been used for decades to introduce traits intoplants. An example of a description of backcrossing (and other plantbreeding methodologies) can be found in, for example, Poelman (1995),supra; and Jensen (1988) Plant Breeding Methodology, Wiley, New York,N.Y. In an exemplary backcross protocol, an original plant of interest(the “recurrent parent”) is crossed to a second plant (the“non-recurrent parent”) that carries the nucleic acid be introduced. Theresulting progeny from this cross are then crossed again to therecurrent parent, and the process is repeated until a converted plant isobtained, wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the nucleic acid from thenon-recurrent parent.

As used herein, the term “isogenic” refers to two individual plants (orportions thereof e.g., seeds, cells) having a substantially identicalgenotype (e.g., not more than 1 gene is different between theindividuals).

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

The terms “plasmid” and “vector,” as used herein, refer to an extrachromosomal element that may carry one or more gene(s) that are not partof the central metabolism of the cell. Plasmids and vectors typicallyare circular double-stranded DNA molecules. However, plasmids andvectors may be linear or circular nucleic acids, of a single- ordouble-stranded DNA or RNA, and may be derived from any source, in whicha number of nucleotide sequences have been joined or recombined into aunique construction that is capable of introducing a promoter fragmentand a coding DNA sequence along with any appropriate 3′ untranslatedsequence into a cell. In examples, plasmids and vectors may compriseautonomously replicating sequences, genome integrating sequences, and/orphage or nucleotide sequences.

Polypeptide and “protein” are used interchangeably herein and include amolecular chain of two or more amino acids linked through peptide bonds.The terms do not refer to a specific length of the product. Thus,“peptides,” and “oligopeptides,” are included within the definition ofpolypeptide. The terms include post-translational modifications of thepolypeptide, for example, glycosylations, acetylations, phosphorylationsand the like. In addition, protein fragments, analogs, mutated orvariant proteins, fusion proteins and the like are included within themeaning of polypeptide. The terms also include molecules in which one ormore amino acid analogs or non-canonical or unnatural amino acids areincluded as can be synthesized, or expressed recombinantly using knownprotein engineering techniques. In addition, inventive fusion proteinscan be derivatized as described herein by well-known organic chemistrytechniques.

The term “fusion protein” indicates that the protein includespolypeptide components derived from more than one parental protein orpolypeptide. Typically, a fusion protein is expressed from a fusion genein which a nucleotide sequence encoding a polypeptide sequence from oneprotein is appended in frame with, and optionally separated by a linkerfrom, a nucleotide sequence encoding a polypeptide sequence from adifferent protein. The fusion gene can then be expressed by arecombinant host cell as a single protein.

IV. Embodiments of the Present Invention

In one embodiment, the subject disclosure relates to an isolatedpolypeptide comprising an amino acid sequence of SEQ ID NO:1. In afurther embodiment, the isolated polypeptide of SEQ ID NO:1 comprises anamino acid sequence which has at least 85% sequence identity to SEQ IDNO:3. In aspects of the embodiment, the isolated polypeptides of SEQ IDNO:1 and SEQ ID NO:3 have cytoplasmic male sterile activity.

In another embodiment, the subject disclosure relates to an isolatedpolypeptide consisting of an amino acid sequence of SEQ ID NO:1. Inanother embodiment, the subject disclosure relates to an isolatedpolypeptide consisting essentially of an amino acid sequence of SEQ IDNO:1. In a further embodiment, the isolated polypeptide of SEQ ID NO:1consists of an amino acid sequence which has at least 85% sequenceidentity to SEQ ID NO:3. In a further embodiment, the isolatedpolypeptide of SEQ ID NO:1 consists essentially of an amino acidsequence which has at least 85% sequence identity to SEQ ID NO:3.

In another embodiment, the disclosure relates to a cell comprising theisolated polypeptide of SEQ ID NO:1. In an aspect, the cell comprisingthe isolated polypeptide of SEQ ID NO:1 can be a plant cell. In furtheraspects, the subject disclosure relates to a plant comprising the plantcell comprising the isolated polypeptide of SEQ ID NO:1. In anotherembodiment, the plant is a monocotyledonous plant. In furtherembodiments, the monocotyledonous plant is a maize plant. In anotherembodiment, the maize plant has CMS-S type cytoplasm. Further aspects ofthe subject disclosure relate to seed obtained from the plant comprisingthe plant cell comprising the isolated polypeptide of SEQ ID NO:1.

In an additional embodiment, the disclosure relates to a syntheticnucleic acid sequence encoding the polypeptide of SEQ ID NO:1. In afurther embodiment, the polypeptide of SEQ ID NO:1 is encoded by thesequence of SEQ ID NO:2. In another embodiment, a polynucleotidecomprises the sequence of SEQ ID NO:2. In another aspect, apolynucleotide consists of the sequence of SEQ ID NO:2. In a furtheraspect, a polynucleotide consists essentially of the sequence of SEQ IDNO:2. In a further aspect the polynucleotide comprising the sequence ofSEQ ID NO:2 is operably linked to a promoter, wherein the promoter isfunctional in plants. In yet another aspect, the promoter is a plantcytoplasmic male sterile promoter. In another embodiment, the disclosurerelates to a cell comprising the polynucleotide that further comprisesthe sequence of SEQ ID NO:2. In another embodiment, the disclosurerelates to a cell consists of the polynucleotide that further comprisesthe sequence of SEQ ID NO:2. In another embodiment, the disclosurerelates to a cell consists essentially of the polynucleotide thatfurther comprises the sequence of SEQ ID NO:2. In a further embodiment,the disclosure relates to a vector comprising the polynucleotide thatfurther comprises the sequence of SEQ ID NO:2. In a further embodiment,the disclosure relates to a vector consisting of the polynucleotide thatfurther comprises the sequence of SEQ ID NO:2. In a further embodiment,the disclosure relates to a vector consisting essentially of thepolynucleotide that further comprises the sequence of SEQ ID NO:2.

Furthermore, the subject disclosure relates to a method for producing aprogeny cytoplasmic male sterile plant, comprising the steps of:

-   -   a) crossing a female parent plant with a male parent plant,        wherein the female parent plant is a cytoplasmic male sterile        parent plant homozygous for SEQ ID NO:4, and wherein the male        parent plant is a fertile parent plant;    -   b) harvesting a progeny seed from the cross of step (a), wherein        the progeny seed is homozygous for SEQ ID NO:4;    -   c) planting the progeny seed; and,    -   d) growing the progeny seed, wherein the progeny seed produce        the progeny cytoplasmic male sterile plant, wherein the progeny        cytoplasmic male sterile plant is homozygous for SEQ ID NO:4.        In a further aspect, the method relates to producing the progeny        cytoplasmic male sterile plant wherein the female and male        parent plants are monocotyledonous plants. In another        embodiment, the monocotyledonous plants are maize plants. In a        further embodiment, the maize plant has CMS-S type cytoplasm. In        yet another embodiment, the male parent plant is isogenic to the        female parent plant. In a subsequent embodiment, the maize        plants are homozygous or heterozygous for SEQ ID NO:8. In        another embodiment, the method further comprises introducing a        desired trait into the progeny cytoplasmic male sterile plant.        In a subsequent embodiment, the desired trait is selected from        the group consisting of an insecticidal resistance trait,        herbicide tolerant trait, disease resistance trait, yield        increase trait, nutritional quality trait, agronomic increase        trait, and combinations thereof.

In yet another aspect, the subject disclosure relates to a method forproducing a progeny cytoplasmic male sterile plant, the method furthercomprising the steps of:

-   -   e) crossing the progeny cytoplasmic male sterile plant, with        another plant comprising a desired trait to produce F1 progeny        plants;    -   f) selecting F1 progeny plants that have the desired trait to        produce selected F1 progeny plants;    -   g) crossing the selected F1 progeny plants with the progeny        cytoplasmic male sterile plant to produce backcross progeny        plants;    -   h) selecting for backcross progeny plants that have the desired        trait; and,    -   i) repeating steps (g) and (h) three or more times in succession        to produce selected fourth or higher backcross progeny plants        that comprise the desired trait.

Further aspects of the subject disclosure relate to a method forrestoring fertility to a progeny of a cytoplasmic male sterile parentplant, the method comprising:

-   -   a) crossing a female parent plant with a male parent plant,        wherein the female parent plant is a cytoplasmic male sterile        parent plant homozygous or heterozygous for SEQ ID NO:8, and        wherein the cytoplasmic male sterile plant is a fertile parent        plant;    -   b) harvesting a progeny seed from the cross of step (a), wherein        the progeny seed is homozygous or heterozygous for SEQ ID NO:8;    -   c) planting the progeny seed;    -   d) growing the progeny seed, wherein the progeny seed produce a        progeny cytoplasmic male fertile plant; and,    -   e) restoring fertility to the progeny of the cytoplasmic male        sterile parent plant, wherein the progeny cytoplasmic male        fertile plant is homozygous or heterozygous for SEQ ID NO:8.        In a further aspect, the method relates to producing the progeny        cytoplasmic male fertile plant wherein the female and male        parent plants are monocotyledonous plants. In another        embodiment, the monocotyledonous plants are maize plants. In yet        another embodiment, the maize plant has CMS-S type cytoplasm. In        a further embodiment, the male parent plant is isogenic to the        female parent plant. In a subsequent embodiment, the maize        plants are homozygous or heterozygous for SEQ ID NO:8. In        another embodiment, the method further comprises introducing a        desired trait into the progeny cytoplasmic male sterile plant.        In another embodiment, the desired trait is selected from the        group consisting of an insecticidal resistance trait, herbicide        tolerant trait, disease resistance trait, yield increase trait,        nutritional quality trait, agronomic increase trait, and        combinations thereof.

In yet another aspect, the subject disclosure relates to a method forproducing a progeny of a cytoplasmic male sterile parent plant, themethod further comprising the steps of:

-   -   f) crossing the progeny cytoplasmic male sterile plant, with        another plant comprising a desired trait to produce F1 progeny        plants;    -   g) selecting F1 progeny plants that have the desired trait to        produce selected F1 progeny plants;    -   h) crossing the selected F1 progeny plants with the progeny        cytoplasmic male sterile plant to produce backcross progeny        plants;    -   i) selecting for backcross progeny plants that have the desired        trait; and,    -   j) repeating steps (g) and (h) three or more times in succession        to produce selected fourth or higher backcross progeny plants        that comprise the desired trait.

In an embodiment, the subject disclosure relates to an isolatedpolypeptide comprising an amino acid sequence of SEQ ID NO:5. In afurther embodiment, the isolated polypeptide of SEQ ID NO:5 comprises anamino acid sequence which has at least 85% sequence identity to SEQ IDNO:7. In aspects of the embodiment, the isolated polypeptides of SEQ IDNO:5 and SEQ ID NO:7 have cytoplasmic male sterile restorer activity.

In an embodiment, the subject disclosure relates to an isolatedpolypeptide consisting of an amino acid sequence of SEQ ID NO:5. In anembodiment, the subject disclosure relates to an isolated polypeptideconsisting essentially of an amino acid sequence of SEQ ID NO:5. In afurther embodiment, the isolated polypeptide of SEQ ID NO:5 consistingof an amino acid sequence which has at least 85% sequence identity toSEQ ID NO:7. In a further embodiment, the isolated polypeptide of SEQ IDNO:5 consisting essentially of an amino acid sequence which has at least85% sequence identity to SEQ ID NO:7.

In another embodiment, the disclosure relates to a cell comprising theisolated polypeptide of SEQ ID NO:5. In an aspect, the cell comprisingthe isolated polypeptide of SEQ ID NO:5 can be a plant cell. In furtheraspects, the subject disclosure relates to a plant comprising the plantcell comprising the isolated polypeptide of SEQ ID NO:5. In anotherembodiment, the plant is a monocotyledonous plant. In furtherembodiments, the monocotyledonous plant is a maize plant. In anotherembodiment, the maize plant has CMS-S type cytoplasm. Further aspects ofthe subject disclosure relate to seed obtained from the plant comprisingthe plant cell comprising the isolated polypeptide of SEQ ID NO:5.

In an additional embodiment, the disclosure relates to a syntheticnucleic acid sequence encoding the polypeptide of SEQ ID NO:5. In afurther embodiment, the synthetic nucleic acid sequence comprises thesequence of SEQ ID NO:5. In another embodiment, the synthetic nucleicacid sequence consists of the sequence of SEQ ID NO:5. In an additionalembodiment, the synthetic nucleic acid sequence consists essentially ofthe sequence of SEQ ID NO:5. In a further embodiment, the syntheticnucleic acid sequence comprises the sequence of SEQ ID NO:8. In anotherembodiment, the synthetic nucleic acid sequence consists of the sequenceof SEQ ID NO:8. In an additional embodiment, the synthetic nucleic acidsequence consists essentially of the sequence of SEQ ID NO:8. In anotherembodiment, a gene expression cassette comprises the synthetic sequenceof SEQ ID NO:8. In a further aspect, the gene expression cassettecomprising the sequence of SEQ ID NO:8 is operably linked to a promoter,wherein the promoter is functional in plants. In yet another aspect, thepromoter is a plant cytoplasmic male sterile promoter. In anotherembodiment, the disclosure relates to a cell comprising the geneexpression cassette that further comprises the sequence of SEQ ID NO:8.In an embodiment, the disclosure relates to a cell comprising the geneexpression cassette that consists of the sequence of SEQ ID NO:8. In anadditional embodiment, the disclosure relates to a cell consistingessentially of the gene expression cassette that further comprises thesequence of SEQ ID NO:8. In a further embodiment, the disclosure relatesto a gene expression cassette comprising the polynucleotide that furthercomprises the sequence of SEQ ID NO:8. In a subsequent embodiment, thedisclosure relates to a gene expression cassette consisting of thepolynucleotide that consists of the sequence of SEQ ID NO:8. In anotherembodiment, the disclosure relates to a gene expression cassettecomprising the polynucleotide that consists essentially of the sequenceof SEQ ID NO:8.

Further aspects of the subject disclosure relate to a method forrestoring fertility to a cytoplasmic male sterile plant, the methodcomprising:

-   -   a) transforming the cytoplasmic male sterile plant with the        synthetic nucleic acid sequence encoding the polypeptide of SEQ        ID NO:5;    -   b) integrating the synthetic nucleic acid sequence into the        genome of the cytoplasmic male sterile plant; and,    -   c) expressing the synthetic nucleic acid sequence, wherein        expression of the synthetic nucleic acid sequence restores        fertility to the cytoplasmic male sterile plant.

Other aspects of the subject disclosure relate to a method for alteringthe morphology of a cytoplasmic male sterile plant, the methodcomprising:

-   -   a) transforming the cytoplasmic male sterile plant with the        synthetic nucleic acid sequence encoding the polypeptide of SEQ        ID NO:5;    -   b) integrating the synthetic nucleic acid sequence into the        genome of the cytoplasmic male sterile plant; and,    -   c) expressing the synthetic nucleic acid sequence, wherein        expression of the synthetic nucleic acid sequence alters the        morphology of the cytoplasmic male sterile plant.

In an embodiment, the subject disclosure relates to a method ofdetecting a plant comprising a cytoplasmic male sterile restorer trait,the method comprising the steps of:

-   -   a) isolating a genomic polynucleotide sample from a plant, plant        tissue, plant part, or plant cell;    -   b) adding a set of oligonucleotide primers to the genomic        polynucleotide sample;    -   c) subjecting the genomic polynucleotide sample and the set of        oligonucleotide primers to an amplification process; and,    -   d) detecting at least one amplified product, wherein the        amplified product indicates the presence of the cytoplasmic male        sterile trait in the plant.        Further embodiments relate to a method for detecting a plant        comprising a cytoplasmic male sterile restorer trait, include        the set of oligonucleotide primers that are selected from the        group consisting of SEQ ID NO:28, SEQ ID NO:29, and SEQ ID        NO:30. In another embodiment, the amplified product comprises        SEQ ID NO:6. In an embodiment, the amplified product consists of        SEQ ID NO:6. In a further embodiment, the amplified product        consists essentially of SEQ ID NO:6. Further embodiments include        the quantitation of the amplified product.

Other aspects of the subject disclosure relate to a method of detectingan expression level of a cytoplasmic male sterile restorer trait, themethod comprising the steps of:

-   -   a) performing a first amplification process using a cytoplasmic        male sterile restorer trait probe (SEQ ID NO:30), a forward        cytoplasmic male sterile restorer trait primer (SEQ ID NO:28)        and a reverse cytoplasmic male sterile restorer trait primer        (SEQ ID NO:29) on a genomic polynucleotide sample, wherein a        first fluorescent dye is released from the cytoplasmic male        sterile restorer trait probe (SEQ ID NO:30);    -   b) performing a second amplification process using a reference        probe (SEQ ID NO:36), a forward reference primer (SEQ ID NO:34)        and a reverse reference primer (SEQ ID NO:35) on the genomic        polynucleotide sample, wherein a second fluorescent dye is        released from the reference probe (SEQ ID NO:36);    -   c) quantitating the relative intensity of the first fluorescent        dye to the second fluorescent dye to detect the expression level        of the cytoplasmic male sterile restorer trait.        Further embodiments relate to the method of detecting an        expression level of a cytoplasmic male sterile restorer trait,        include where the first and second amplification processes are        performed in a single PCR assay tube using probes with different        fluorescent dyes.

In a further embodiment, the method of detecting an expression level ofa cytoplasmic male sterile restorer trait comprises the steps of;

-   -   d) loading a PCR solution in a single PCR assay tube, the PCR        solution comprising a polymerase with 5′ to 3′ nuclease        activity, deoxynucleotides, the primers, the probes, a buffer,        and the genomic polynucleotide sample;    -   e) amplifying the PCR solution, wherein the PCR solution is        treated under amplification conditions such that the 5′ to 3′        nuclease activity of the polymerase cleaves the probes thereby        releasing the fluorescent dye that emits light; and,    -   f) measuring the light emitted from fluorescent dye, during the        amplification.        Further embodiments relate to the method of detecting an        expression level of a cytoplasmic male sterile restorer trait,        include the cytoplasmic male sterile restorer trait probe        comprising the first fluorescent dye and a first quencher. In a        subsequent embodiment, the first fluorescent dye is selected        from the group consisting of a HEX fluorescent dye, a VIC        fluorescent dye, a FAM fluorescent dye, a JOE fluorescent dye, a        TET fluorescent dye, a Cy 3 fluorescent dye, a Cy 3.5        fluorescent dye, a Cy 5 fluorescent dye, a Cy 5.5 fluorescent        dye, a Cy 7 fluorescent dye, or a ROX fluorescent dye. In a        further embodiment, the first quencher is selected from the        group consisting of a Dabcyl quencher, a Tamra quencher, a Qxl        quencher, an Iowa Black FQ quencher, an Iowa Black RQ quencher,        an IR Dye QC-1 quencher, a MGB quencher, or a Blackhole        quencher. In yet another embodiment, the reference probe        comprises the second fluorescent dye and a second quencher. In a        subsequent embodiment, the second fluorescent dye is selected        from the group consisting of a HEX fluorescent dye, a FAM        fluorescent dye, a VIC fluorescent dye, a JOE fluorescent dye, a        TET fluorescent dye, a Cy 3 fluorescent dye, a Cy 3.5        fluorescent dye, a Cy 5 fluorescent dye, a Cy 5.5 fluorescent        dye, a Cy 7 fluorescent dye, or a ROX fluorescent dye. In        another embodiment, the second quencher is selected from the        group consisting of a Dabcyl quencher, a Tamra quencher, a Qxl        quencher, an Iowa Black FQ quencher, an Iowa Black RQ quencher,        an IR Dye QC-1 quencher, a MGB quencher, or a Blackhole        quencher. In a further embodiment, the genomic polynucleotide        sample comprises cDNA produced from mRNA isolated from a plant,        plant tissue, plant part, or plant cell.

In one embodiment of the present disclosure, a polynucleotide donorcassette comprising an isolated polypeptide comprising an amino acidsequence of SEQ ID NO: 1 is provided. In a further embodiment, theisolated polypeptide of SEQ ID NO:1 further comprises an amino acidsequence with at least 85% sequence identity to SEQ ID NO:3. In asubsequent embodiment, the isolated polypeptide of SEQ ID NO:1 and apolypeptide with 85% sequence identity to SEQ ID NO:3 have cytoplasmicmale sterile activity.

Cytoplasmic male sterile systems have been used to breed hybrids in anumber of crop species. The use of such a sterility system can becost-effective and labor conscious. In maize or corn, for example, theexpensive and laborious task of detasselling is avoided when cytoplasmicmale sterility is utilized to avoid self-pollinating. The use of thecytoplasmic male sterile system in a breeding program is alsoadvantageous because of its simplicity and cost savings.

Cytoplasmic male sterility, a maternally inherited trait, is most widelyused in the hybrid industry to render the male properties of a plantnonfunctional. During fertilization the female contributes a haploidnucleus and virtually all of the cytoplasm from the egg, while the malecontributes a haploid nucleus but almost no cytoplasm from the pollen.The result being that the female cytoplasm which confers male sterilityis passed from generation to generation. Information carried in thecytoplasm affecting the phenotype, i.e., anther presence or pollenproduction is contributed exclusively by the female. This type ofsterility affects only pollen production; seed set is normal. Generally,all the progeny from a male sterile plant are themselves male sterile.However, in some cases male fertility can be restored. Pearson, O. H.(1981) Hort Sci. 16: 482-487. Fertility can be restored either bycytoplasmic reversion to fertility or by a nuclear restorer gene able tooverride the effects of cytoplasm. MacKenzie, S. A. et al. (1988) Proc.Natl. Acad. Sci. USA J35: 2714-2717. A specific cytoplasm can be carriedalong from generation to generation provided the plant possessing thecytoplasm is the maternal parent in each cross.

Typically, upon identification of a source of cytoplasmic malesterility, the “rf” trait is transferred to a desirable “female” or “A”line. A “maintenance” or “B” line lacking both the sterility trait andrestoration factor is used to perpetuate and increase the female line. A“restorer” or “Rf” line, carrying a pollen fertility factor is used as amale to pollinate the cytoplasmic male sterile “A” line to create ahybrid variety. The cytoplasmic male sterile plant of the “A” line canbe crossed with a plant from a different variety to produce hybridprogeny. This type of breeding program is often referred to as acytoplasmic male sterile-restorer system.

Various Rf alleles have been sequences or cloned, for example, Rf2 frommaize (Zea mays) (Cui, X., Wise, R. P., and Schnable, P. S. 1996. TheRf2 nuclear restorer gene of male-sterile T-cytoplasm maize. Science272:1334-1336), Rf-PPR592 from Petunia (Petunia hybrida) (Bentolila, S.,Alfonso, A. A., and Hanson, M. R. 2002. A pentatricopeptiderepeat-containing gene restores fertility to male sterile plants. Proc.Natl. Acad. Sci. USA 99:10887-10892), Rfo from radish (Raphanus sativus)(Brown, G. G., Formanova, N., Jin, H., Wargachuk, R., Dendy, C., Patil,P., Laforest, M., Zhang, J. F., Cheung, W. Y., and Landry, B. S. 2003.The radish Rf Restorer gene of Ogura cytoplasmic male sterility encodesa protein with multiple pentatricopeptide repeats. Plant J. 35:262-272;Desloire, S., Gherbi, H., Laloui, W., Marhadour, S., Clouet, V.,Cattolico, L., Falentin, C., Giancola, S., Renard, M., Burdar, F.,Small, I., Caboche, M., and Bendahmane, A. 2003. Identification of thefertility restorer locus, Rfo, in radish, as a member of thepentatricopeptide-repeat protein family. EMBO Rep. 4:1-7; Koizuka, N.,Imai, R., Fujimoto, H., Hayakawa, T., Kimura, Y., Kohno-Murase, J.,Sakai, T., Kawasaki, S., Imamura, J. 2003. genetic characterization of apentatricopeptide repeat protein gene, orf 687, that restores fertilityin the cytoplasmic male sterile Kosena radish. Plant J. 34:407-415), Rf1and Rf2 from sorghum (Sorghum bicolor) (Klein, R. R., Klein, P. E.,Mullet, J. E., Minx, P., Rooney, W. L., and Schertz, K. F. 2005.Fertility restorer locus Rf1 of sorghum (Sorghum bicolor L.) encodes apentatricopeptide repeat protein not present in the collinear region ofrice chromosome 12. Theor. A. 1. Genet. 111:994-1012), Rf1a and Rf1bfrom rice (Oryza sativa) for BT-type CMS (Kazama, T., and Toriyama, K.2003. A pentatricopeptide repeat-containing gene that promotes theprocessing of aberrant atp6 RNA of cytoplasmic male-sterile rice. FEBSLett. 544:99-102; Akagi, H., Nakamura, A., Yokozeki-Misono, Y., Inagaki,A., Takahashi, H., Mori, K., and Fujimura, T. 2004. Position cloning ofthe rice Rf-1 gene, a restorer of BT-type cytoplasmic male sterilitythat encodes a mitochondria-targeting PPR protein. Theor. Appl. Genet.108:1449-1457; Komori, T., Ohta, /s., Murai, N., Takakura, Y., Kuraya,Y., Suzuki, S., Hiei, Y., Imaseki, H., and Nitta, N. 2004. Map-basedcloning of a fertility restorer gene, Rf-1 in rice (Oryza sativa L.).Plant J. 37:315-325; and, Wang, Z., Zou, Y., Li, X., Zhang, Q., Chen,L., Wu, H., Su, D., Chen, Y., Guo, J., Luo, D., Long, Y., Zhong, Y., andLiu, Y. G. 2006b. Cytoplasmic male sterility of rice with Boro IIcytoplasm is caused by a cytotoxic peptide and is restored by tworelated PPR motif genes via distinct modes of mRNA silencing. Plant Cell18:676-687), Rf17 (RMS) from rice (Oryza sativa) for CW-type CMS (FujiiS. & Toriyama K. 2009. Suppressed expression of RETROGRADE-REGULATEDMALE STERILITY restores pollen fertility in cytoplasmic male sterilerice plants. PNAS 106:9513-9518), Rf1 & Rf2 from monkey flower (Mimulusguttatus) (Barr, C. M., & Fishman L. 2010. The Nuclear component of acytonuclear hybrid incompatibility in Mimulus maps to a cluster ofpentatricopeptide repeat genes. Genetics 184:455-465), Rf2 for LeadRice-type CMS from rice (Oryza sativa) (Itabashi, E., Iwata, N., Fujii,S., Kazama, T. and Toriyama, K. 2011. The Fertility restorer gene, Rf2for lead Rice-type cytoplasmic male sterility of rice encodes amitochondrial glycine-rich protein. The plant Journal (2011)65:359-367), Rf5 for Hong-Lian CMS from rice (Oryza sativa) (Hu, J.,Wang, K., Huang, W., Liu, G., Wang, J., Huang, Q., Ji, Y., Qin, X., Wan,L., Zhu, R., Li, S., Yang, D. and Zhu, Y. 2012. The RicePentatricopeptide Repeat Protein Rf5 Restores Fertility in Hong-LianCytoplasmic Male-Sterile Lines via a Complex with the Glycine-RichProtein GRP162. The plant cell advance online publication 2012), and Rf4for CMS C-type of maize (Zea mays) (U.S. Patent Pub. No. 2012/0090047A1).

In other embodiments the isolated polypeptide of SEQ ID NO:1 furthercomprises an amino acid sequence with at least 85%, 87.5%, 90%, 92.5%,95%, 97.5%, or 99% sequence identity to SEQ ID NO:3. In a subsequentembodiment, the isolated polypeptide of SEQ ID NO:1 and a polypeptidewith 85%, 87.5%, 90%, 92.5%, 95%, 97.5% or 99% sequence identity to SEQID NO:3 have cytoplasmic male sterile activity.

The term “percent identity” (or “% identity”), as known in the art, is arelationship between two or more polypeptide sequences or two or morepolynucleotide sequences, as determined by comparing the sequences. Inthe art, “identity” also means the degree of sequence relatednessbetween polypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those disclosed in: 1.) Computational MolecularBiology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991).

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100. See Russell, R.,and Barton, G., “Structural Features can be Unconserved in Proteins withSimilar Folds,” J. Mol. Biol. 244, 332-350 (1994), at p. 337, which isincorporated herein by reference in its entirety.

In addition, methods to determine identity and similarity are codifiedin publicly available computer programs. Sequence alignments and percentidentity calculations can be performed, for example, using the AlignXprogram of the Vector NTI® suite (Invitrogen, Carlsbad, Calif.) orMegAlign™ program of the LASERGENE bioinformatics computing suite(DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences isperformed using the “Clustal method of alignment” which encompassesseveral varieties of the algorithm including the “Clustal V method ofalignment” corresponding to the alignment method labeled Clustal V(disclosed by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in theMegAlign™ program of the LASERGENE bioinformatics computing suite(DNASTAR Inc.). For multiple alignments, the default values correspondto GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters forpairwise alignments and calculation of percent identity of proteinsequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2,GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of thesequences using the Clustal V program, it is possible to obtain a“percent identity” by viewing the “sequence distances” table in the sameprogram. Additionally the “Clustal W method of alignment” is availableand corresponds to the alignment method labeled Clustal W (described byHiggins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al.,Comput. Appl. Biosci. 8:189-191(1992)) and found in the MegAlign™ v6.1program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.).Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTHPENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5,Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). Afteralignment of the sequences using the Clustal W program, it is possibleto obtain a “percent identity” by viewing the “sequence distances” tablein the same program.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides, from otherspecies, wherein such polypeptides have the same or similar function oractivity. Useful examples of percent identities include, but are notlimited to: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or anyinteger percentage from 55% to 100% may be useful in describingembodiments of the present disclosure, such as 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Suitablenucleic acid fragments not only have the above homologies but typicallyencode a polypeptide having at least 50 amino acids, preferably at least100 amino acids, more preferably at least 150 amino acids, still morepreferably at least 200 amino acids, and most preferably at least 250amino acids.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.,215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

When referring to hybridization techniques, all or part of a knownnucleotide sequence can be used as a probe that selectively hybridizesto other corresponding nucleotide sequences present in a population ofcloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNAlibraries) from a chosen organism. The hybridization probes may begenomic DNA fragments, plasmid DNA fragments, cDNA fragments, RNAfragments, PCR amplified DNA fragments, oligonucleotides, or otherpolynucleotides, and may be labeled with a detectable group such as ³²P,or any other detectable marker. Thus, for example, probes forhybridization can be made by labeling synthetic oligonucleotides basedon the DNA sequences of embodiments of the disclosure. Methods forpreparation of probes for hybridization and for construction of cDNA andgenomic libraries are generally known in the art and are disclosed(Sambrook et al., 1989).

The nucleic acid probes and primers of embodiments of the presentdisclosure hybridize under stringent conditions to a target DNAsequence. Any conventional nucleic acid hybridization or amplificationmethod can be used to identify the presence of DNA from a transgenicevent in a sample. Nucleic acid molecules or fragments thereof arecapable of specifically hybridizing to other nucleic acid moleculesunder certain circumstances. As used herein, two nucleic acid moleculesare said to be capable of specifically hybridizing to one another if thetwo molecules are capable of forming an anti-parallel, double-strandednucleic acid structure. A nucleic acid molecule is said to be the“complement” of another nucleic acid molecule if the two nucleic acidmolecules exhibit complete complementarity. As used herein, moleculesare said to exhibit “complete complementarity” when every nucleotide ofone of the molecules is complementary to a nucleotide of the other.Molecules that exhibit complete complementarity will generally hybridizeto one another with sufficient stability to permit them to remainannealed to one another under conventional “high-stringency” conditions.Conventional high-stringency conditions are described by Sambrook etal., 1989.

Two molecules are said to exhibit “minimal complementarity” if they canhybridize to one another with sufficient stability to permit them toremain annealed to one another under at least conventional“low-stringency” conditions. Conventional low-stringency conditions aredescribed by Sambrook et al., 1989. In order for a nucleic acid moleculeto serve as a primer or probe, it need only exhibit the minimalcomplementarity of sequence to be able to form a stable double-strandedstructure under the particular solvent and salt concentrations employed.

Factors that affect the stringency of hybridization are well-known tothose of skill in the art and include, but are not limited to,temperature, pH, ionic strength, and concentration of organic solventssuch as, for example, formamide and dimethylsulfoxide. As is known tothose of skill in the art, hybridization stringency is increased byhigher temperatures, lower ionic strength and lower solventconcentrations.

The term “stringent condition” or “stringency conditions” isfunctionally defined with regard to the hybridization of a nucleic-acidprobe to a target nucleic acid (i.e., to a particular nucleic-acidsequence of interest) by the specific hybridization procedure discussedin Sambrook et al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989at 9.47-9.52 and 9.56-9.58.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na⁺ ion, typically about 0.01 to1.0 M Na⁺ ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1.0 M NaCl, 0.1% SDS (sodium dodecyl sulfate) at 37°C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodiumcitrate) at 50 to 55° C. Exemplary moderate stringency conditionsinclude hybridization in 40 to 45% formamide, 1.0 M NaCl, 0.1% SDS at37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary highstringency conditions include hybridization in 50% formamide, 1.0 MNaCl, 0.1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically a function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation T_(m)=81.5° C.+16.6 (log M)+0.41(% GC)-0.61(% form.)-500/L,where M is the molarity of monovalent cations, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % form. is the percentageof formamide in the hybridization solution, and L is the length of thehybrid in base pairs (Meinkoth and Wahl, 1984). The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of acomplementary target sequence hybridizes to a perfectly matched probe.T_(m) is reduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization, and/or wash conditions can be adjusted for sequences ofthe desired identity to hybridize. For example, if sequences with 90%identity are sought, the T_(m) can be decreased 10° C. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (T_(m)); lowstringency conditions can utilize a hybridization and/or wash at 11 to20° C. lower than the thermal melting point (T_(m)). Using the equation,hybridization and wash compositions, and desired T_(m), those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T_(m) of less than 45° C.(aqueous solution) or 32° C. (formamide solution), it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found (1997)Ausubel et al, Short Protocols in Molecular Biology, pages 2-40, ThirdEdit. (1997) and Sambrook et al. (1989).

In other embodiments, the subject disclosure provides a cell comprisingthe polypeptide of SEQ ID NO:1 or SEQ ID NO:3. The term “cell” asreferred to herein encompasses a living organism capable of selfreplication, and may be a eukaryotic or prokaryotic cell. In someembodiments the cell is a plant cell. In some embodiments, the plantcell can be but is not limited to any higher plant, including bothdicotyledonous and monocotyledonous plants, and consumable plants,including crop plants and plants used for their oils. Thus, any plantspecies or plant cell can be selected as described further below.

In some embodiments, plant cells in accordance with the presentdisclosure includes, but is not limited to, any higher plants, includingboth dicotyledonous and monocotyledonous plants, and particularlyconsumable plants, including crop plants. Such plants can include, butare not limited to, for example: alfalfa, soybeans, cotton, rapeseed(also described as canola), linseed, corn, rice, brachiaria, wheat,safflowers, sorghum, sugarbeet, sunflowers, tobacco and turf grasses.Thus, any plant species or plant cell can be selected. In embodiments,plant cells used herein, and plants grown or derived therefrom, include,but are not limited to, cells obtainable from rapeseed (Brassica napus);indian mustard (Brassica juncea); Ethiopian mustard (Brassica carinata);turnip (Brassica rapa); cabbage (Brassica oleracea); soybean (Glycinemax); linseed/flax (Linum usitatissimum); maize (also described as corn)(Zea mays); safflower (Carthamus tinctorius); sunflower (Helianthusannuus); tobacco (Nicotiana tabacum); Arabidopsis thaliana; Brazil nut(Betholettia excelsa); castor bean (Ricinus communis); coconut (Cocusnucifera); coriander (Coriandrum sativum); cotton (Gossypium spp.);groundnut (Arachis hypogaea); jojoba (Simmondsia chinensis); oil palm(Elaeis guineeis); olive (Olea eurpaea); rice (Oryza sativa); squash(Cucurbita maxima); barley (Hordeum vulgare); sugarcane (Saccharumofficinarum); rice (Oryza sativa); wheat (Triticum spp. includingTriticum durum and Triticum aestivum); and duckweed (Lemnaceae sp.). Insome embodiments, the genetic background within a plant species mayvary.

In a further embodiment, the subject disclosure provides a seedcomprising the polypeptide of SEQ ID NO:1 or SEQ ID NO:3. In subsequentembodiments a seed from maize is provided. A maize seed may be describedor referred to as a kernel, and is composed of four structural parts:(1) the pericarp, which is a protective outer covering (also known asbran or hull); (2) the germ (also known as an embryo); (3) theendosperm; and, (4) the tip cap, which is the point of attachment to thecob. Another aspect of the present disclosure is one or more parts ofcorn seed, such as the pericarp of the corn seed or the germ and/or theendosperm of the corn seed which remain upon removal of the pericarp andadhering remnants of the seed coat.

The subject disclosure also relates to one or more corn plant parts ofan rf3 or Rf3 corn line. Corn plant parts include plant cells, plantprotoplasts, plant cell tissue cultures from which corn plants can beregenerated, plant DNA, plant calli, plant clumps, and plant cells thatare intact in plants or parts of plants, such as embryos, pollen,ovules, flowers, seeds, kernels, ears, cobs, leaves, husks, stalks,roots, root tips, brace roots, lateral tassel branches, anthers,tassels, glumes, silks, tillers, and the like.

In subsequent embodiments, the subject disclosure relates to a cellcomprising SEQ ID NO:5. In other embodiments, the cell is a plant cell.Further embodiments include a plant comprising the plant cell. In someembodiments the plant may be a monocotyledonous or dicotyledonous plant.In other embodiments, the monocotyledonous plant is a maize plant.Further embodiments include a maize plant with CMS-S type cytoplasm.Additional embodiments include a plant part, plant tissue, or plantseed.

V. Polynucleotide Sequences Encoding the Isolated Protein

The subject disclosure further provides a synthetic nucleic acidsequence encoding the polypeptide of SEQ ID NO:1 or SEQ ID NO:3. In anembodiment, the synthetic nucleic acid sequence comprising thepolypeptide of SEQ ID NO:1 is encoded by SEQ ID NO:2. In furtherembodiments, the synthetic nucleic acid sequence comprising thepolypeptide of SEQ ID NO:3 is encoded by SEQ ID NO:4. In subsequentembodiments, a sequence with 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, or 99%sequence identify of SEQ ID NO:4 is provided. In other embodiments, apolynucleotide comprising the synthetic nucleic acid sequence of SEQ IDNO:2. In an embodiment, the polynucleotide sequences that comprise thesynthetic nucleic acid sequence of SEQ ID NO:2, may include a geneexpression cassette, a vector, a plasmid, a bacterial artificialchromosome, a DNA fragment, an oligonucleotide, a primer, or a probe.

In further embodiments, the synthetic nucleic acid sequence of SEQ IDNO:2 is operably linked to a promoter, wherein the promoter isfunctional in plants. In an aspect of the embodiment, the promoter is aplant cytoplasmic male sterile promoter

In an embodiment the synthetic nucleic acid sequence of SEQ ID NO:2 isoperably linked to a promoter. The promoter used to direct expression ofa peptide encoding nucleic acid depends on the particular application.For example, a number of promoters that direct expression of a gene in aplant can be employed. Such promoters can be selected from constitutive,chemically-regulated, inducible, tissue-specific, and seed-preferredpromoters.

The range of available plant compatible promoters includes tissuespecific and inducible promoters. An inducible regulatory element is onethat is capable of directly or indirectly activating transcription ofone or more DNA sequences or genes in response to an inducer. In theabsence of an inducer the DNA sequences or genes will not betranscribed. Typically the protein factor that binds specifically to aninducible regulatory element to activate transcription is present in aninactive form which is then directly or indirectly converted to theactive form by the inducer. The inducer can be a chemical agent such asa protein, metabolite, growth regulator, herbicide or phenolic compoundor a physiological stress imposed directly by heat, cold, salt, or toxicelements or indirectly through the action of a pathogen or disease agentsuch as a virus. Typically the protein factor that binds specifically toan inducible regulatory element to activate transcription is present inan inactive form which is then directly or indirectly converted to theactive form by the inducer. The inducer can be a chemical agent such asa protein, metabolite, growth regulator, herbicide or phenolic compoundor a physiological stress imposed directly by heat, cold, salt, or toxicelements or indirectly through the action of a pathogen or disease agentsuch as a virus. A plant cell containing an inducible regulatory elementmay be exposed to an inducer by externally applying the inducer to thecell or plant such as by spraying, watering, heating or similar methods.In some embodiments the synthetic nucleic acid sequence that is operablylinked to a promoter may further comprise a coding sequence for atransit peptide. Examples of transit peptides are known in the art andinclude mitochondrial or chloroplast targeting transit peptides.

Subsequent embodiments of the subject disclosure relate to a cellcomprising the synthetic nucleic acid sequence of SEQ ID NO:2.Additional embodiments of the subject disclosure relate to a cellcomprising the synthetic nucleic acid sequence of SEQ ID NO:4. Furtherembodiments of the subject disclosure relate to a vector comprising thesynthetic nucleic acid sequence of SEQ ID NO:2. Additional embodimentsof the subject disclosure relate to a cell comprising the syntheticnucleic acid sequence of SEQ ID NO:4. In other embodiments the subjectdisclosure provides a cell comprising any previous embodiment of saidvector.

In other embodiments the vector comprises a plasmid. A plasmid or vectorcan be described or referred to as prokaryotic vectors, shuttle vectors,insect vectors, or eukaryotic vectors. Typically, plasmids areextra-chromosomal elements, often circular DNA, that are comprised of anorigin of replication and a selectable marker gene. At times, it may bepreferable to have a plasmid that is functional in E. coli (e.g., DNAsequence analysis, construction of inserts, obtaining quantities ofnucleic acids). The particular plasmid can be selected with regard tothe intended use (e.g., expression in plants, animals, bacteria, fungus,and protozoa). Standard bacterial, plant, and animal expression plasmidsare known in the art and are described in detail, for example, U.S.Patent Publication 20050064474A1 and International Patent PublicationsWO 05/084190, WO05/014791 and WO03/080809. Examples of a plasmid includea pUC19 plasmid. pUC19 is a small double stranded DNA circle. Theplasmid contains high copy number origin of replication that is capableof bacterial replication and contains a multiple cloning site. SeeYanisch-Perron, C., Vieira, J. and Messing, J. (1985). Gene. 33,103-119. Other plasmids are known and commonly used in the art. Forexample, pUC18, pBR322, pBR325, pBR328, pACYC184, pAT153, pUC118, andpUC119 are plasmids commonly known in the art.

In further embodiments of the subject disclosure, an isolatedpolypeptide comprising an amino acid sequence of SEQ ID NO: 5 isprovided. In a further embodiment, the isolated polypeptide of SEQ IDNO:5 further comprises an amino acid sequence with at least 85% sequenceidentity to SEQ ID NO:7. In a subsequent embodiment, the isolatedpolypeptide of SEQ ID NO:5 and a polypeptide with 85% sequence identityto SEQ ID NO:7 have cytoplasmic male sterile activity.

The subject disclosure further provides a synthetic nucleic acidsequence encoding the polypeptide of SEQ ID NO:5 or SEQ ID NO:7. In anembodiment, the synthetic nucleic acid sequence comprising thepolypeptide of SEQ ID NO:5 is encoded by SEQ ID NO:6. In furtherembodiments, the synthetic nucleic acid sequence comprising thepolypeptide of SEQ ID NO:7 is encoded by SEQ ID NO:8. In subsequentembodiments, a sequence with 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, or 99%sequence identify of SEQ ID NO:8 is provided. In other embodiments, apolynucleotide comprising the synthetic nucleic acid sequence of SEQ IDNO:8. In an embodiment, the polynucleotide sequences that comprise thesynthetic nucleic acid sequence of SEQ ID NO:8, may include a geneexpression cassette, a vector, a plasmid, a bacterial artificialchromosome, a DNA fragment, an oligonucleotide, a primer, or a probe.

In other embodiments, the subject disclosure relates to a syntheticnucleic acid sequence encoding the polypeptide of SEQ ID NO:5. Infurther embodiments, the synthetic nucleic acid sequence has thesequence of SEQ ID NO:8. In additional embodiments, a gene expressioncassette comprising the synthetic nucleic acid sequence of SEQ ID NO:8is provided. Further embodiments include the gene expression cassettecomprising the synthetic nucleic acid sequence of SEQ ID NO:8 operablylinked to a promoter, wherein the promoter is functional in plants. Inadditional embodiments the promoter is a cytoplasmic male sterilepromoter. Additional embodiments include a cell comprising the geneexpression cassette comprising the synthetic nucleic acid sequence ofSEQ ID NO:8. Further embodiments of the subject disclosure relate to avector comprising the synthetic nucleic acid sequence of SEQ ID NO:8. Inother embodiments the subject disclosure provides a cell comprising saidvector.

VI. Introgression of RF3 Alleles into Progeny Plants

An embodiment of the subject disclosure provides a method for producinga progeny cytoplasmic male sterile plant, the method comprising thesteps of:

-   -   a) crossing a female parent plant with a male parent plant,        wherein the female parent plant is a cytoplasmic male sterile        parent plant homozygous for SEQ ID NO:4, and wherein the male        parent plant is a fertile parent plant;    -   b) harvesting a progeny seed from the cross of (a), wherein the        progeny seed is homozygous for SEQ ID NO:4;    -   c) planting the progeny seed; and,    -   d) growing the progeny seed, wherein the progeny seed produce        the progeny cytoplasmic male sterile plant, wherein the progeny        cytoplasmic male sterile plant is homozygous for SEQ ID NO:4.

In additional embodiments, the subject disclosure relates to female andmale parent plants that are monocotyledonous plants. In otherembodiments, the monocotyledonous plants are maize plants. In furtherembodiments the male parent plant is isogenic to the female parentplant. In an aspect of the embodiment, the male parent plant ishomozygous or heterozygous for SEQ ID NO:8.

In yet another aspect of the subject disclosure, processes are providedfor producing progeny plants, which processes generally comprisecrossing a first parent corn plant with a second parent corn plantwherein at least one of the first parent corn plant or the second parentcorn plant is a cytoplasmic male sterile plant having CMS-S typecytoplasm. In some embodiments of the present disclosure, thecytoplasmic male sterile plant is a female and in other embodiments thecytoplasmic male restorer plant is a male. These processes may befurther exemplified as processes for producing progeny seed or plants,wherein a first corn plant is crossed with a second corn plant.

Any time the cytoplasmic male sterile plant or the cytoplasmic malerestorer plant is crossed with another, different corn inbred, a progenyor first generation (F₁) corn hybrid plant is produced. As such, aprogeny or F₁ hybrid corn plant may be produced by crossing thecytoplasmic male sterile plant or the cytoplasmic male restorer plantwith any second inbred corn plant. Therefore, any progeny or F₁ hybridcorn plant or corn seed which is produced with the cytoplasmic malesterile plant or the cytoplasmic male restorer plant as a parent is anembodiment of the subject disclosure.

When the cytoplasmic male sterile plant or the cytoplasmic male restorerplant is crossed with another inbred plant to yield a progeny or hybrid,the original parent can serve as either the maternal or paternal plantwith basically, the same characteristics in the hybrids. Occasionally,maternally inherited characteristics may express differently dependingon the decision of which parent to use as the female. However, often oneof the parental plants is preferred as the maternal plant because ofincreased seed yield and preferred production characteristics, such asoptimal seed size and quality or ease of tassel removal. Some plantsproduce tighter ear husks leading to more loss, for example due to rot,or the ear husk may be so tight that the silk cannot completely push outof the tip preventing complete pollination resulting in lower seedyields. There can be delays in silk formation which deleteriously affecttiming of the reproductive cycle for a pair of parental inbreds. Seedcoat characteristics can be preferable in one plant which may affectshelf life of the hybrid seed product. Pollen can shed better by oneplant, thus rendering that plant as the preferred male parent.

In embodiments of the present disclosure, the first step of “crossing”the cytoplasmic male sterile plant or the cytoplasmic male restorerplant comprises planting, preferably in pollinating proximity, seeds ofa first inbred corn plant and a second, distinct inbred corn plant.

A further step comprises cultivating or growing the seeds of thecytoplasmic male sterile plant or the cytoplasmic male restorer plantthat bear flowers. If the parental plants differ in timing of sexualmaturity, techniques may be employed to obtain an appropriate nick,i.e., to ensure the availability of pollen from the parent corn plantdesignated the male during the time at which silks on the parent cornplant designated the female are receptive to the pollen. Methods thatmay be employed to obtain the desired nick include delaying theflowering of the faster maturing plant, such as, but not limited todelaying the planting of the faster maturing seed, cutting or burningthe top leaves of the faster maturing plant (without killing the plant)or speeding up the flowering of the slower maturing plant, such as bycovering the slower maturing plant with film designed to speedgermination and growth or by cutting the tip of a young ear shoot toexpose silk.

In an embodiment, the male sterile plant or the cytoplasmic malerestorer plant are treated with one or more agricultural chemicals asconsidered appropriate by the grower.

A further step comprises harvesting the seeds, near or at maturity, fromthe ear of the plant that received the pollen. In a particularembodiment, seed is harvested from the female parent plant, and whendesired, the harvested seed can be grown to produce a progeny or firstgeneration (F₁) hybrid corn plant.

Yet another step comprises drying and conditioning the seeds, includingthe treating, sizing (or grading) of seeds, and packaging for sale togrowers for the production of grain or forage. As with inbred seed, itmay be desirable to treat hybrid seeds with compositions that render theseeds and seedlings grown therefrom more hardy when exposed to adverseconditions. Mention should be made that resulting progeny or hybrid seedmay be sold to growers for the production of grain and forage and notfor breeding or seed production.

Still further, the subject disclosure provides a progeny corn plantproduced by growing the harvested seeds produced on the cytoplasmicmale-sterile or restorer plant as well as grain produced by the progenycorn plant.

In a subsequent embodiment, the disclosure related to introducing adesired trait into the progeny cytoplasmic male sterile plant. In anaspect of the embodiment, the desired trait is selected from the groupconsisting of an insecticidal resistance trait, herbicide toleranttrait, disease resistance trait, yield increase trait, nutritionalquality trait, agronomic increase trait, and combinations thereof. Otherexamples of a desired trait include modified fatty acid metabolism, forexample, by transforming a plant with an antisense gene of stearoyl-ACPdesaturase to increase stearic acid content of the plant. See Knultzonet al., Proc. Natl. Acad. Sci. USA 89: 2624 (1992). Decreased phytatecontent: (i) Introduction of a phytase-encoding gene would enhancebreakdown of phytate, adding more free phosphate to the transformedplant. For example, see Van Hartingsveldt et al., Gene 127: 87 (1993),for a disclosure of the nucleotide sequence of an Aspergillus nigerphytase gene. (ii) A gene could be introduced that reduces phytatecontent. In corn, this, for example, could be accomplished, by cloningand then reintroducing DNA associated with the single allele which isresponsible for corn mutants characterized by low levels of phytic acid.See Raboy et al., Maydica 35: 383 (1990). (iii) Modified carbohydratecomposition effected, for example, by transforming plants with a genecoding for an enzyme that alters the branching pattern of starch. SeeShiroza et al., J. Bacteriol. 170: 810 (1988) (nucleotide sequence ofStreptococcus mutans fructosyltransferase gene), Steinmetz et al., Mol.Gen. Genet. 200: 220 (1985) (nucleotide sequence of Bacillus subtilluslevansucrase gene), Pen et al., Bio/Technology 10: 292 (1992)(production of transgenic plants that express Bacillus licheniformisα-amylase), Elliot et al., Plant Molec. Biol. 21: 515 (1993) (nucleotidesequences of tomato invertase genes), Sogaard et al., J. Biol. Chem.268: 22480 (1993) (site-directed mutagenesis of barley α-amylase gene),and Fisher et al., Plant Physiol. 102: 1045 (1993) (corn endospermstarch branching enzyme II). Further examples of potentially desiredcharacteristics include greater yield, better stalks, better roots,reduced time to crop maturity, better agronomic quality, highernutritional value, higher starch extractability or starchfermentability, resistance and/or tolerance to insecticides, herbicides,pests, heat and drought, and disease, and uniformity in germinationtimes, stand establishment, growth rate, maturity and kernel size.

In an additional embodiment, the subject disclosure relates to a methodfor producing a progeny cytoplasmic male sterile plant, the methodfurther comprising the steps of:

-   -   e) crossing the progeny cytoplasmic male sterile plant, with        another plant comprising a desired trait to produce F1 progeny        plants;    -   f) selecting F1 progeny plants that have the desired trait to        produce selected F1 progeny plants;    -   g) crossing the selected F1 progeny plants with the progeny        cytoplasmic male sterile plant to produce backcross progeny        plants;    -   h) selecting for backcross progeny plants that have the desired        trait; and,    -   i) repeating steps (g) and (h) three or more times in succession        to produce selected fourth or higher backcross progeny plants        that comprise the desired trait.

Various breeding schemes may be used to produce progeny plants. In onemethod, generally referred to as the pedigree method, the parent may becrossed with another different plant such as a second inbred parent cornplant, which either itself exhibits one or more selected desirablecharacteristic(s) or imparts selected desirable characteristic(s) to ahybrid combination. If the two original parent corn plants do notprovide all the desired characteristics, then other sources can beincluded in the breeding population. Progeny plants, that is, purebreeding, homozygous inbred lines, can also be used as startingmaterials for breeding or source populations from which to developprogeny plants.

Thereafter, resulting seed is harvested and resulting superior progenyplants are selected and selfed or sib-mated in succeeding generations,such as for about 5 to about 7 or more generations, until a generationis produced that no longer segregates for substantially all factors forwhich the inbred parents differ, thereby providing a large number ofdistinct, pure-breeding inbred lines.

In another embodiment for generating progeny plants, generally referredto as backcrossing, one or more desired traits may be introduced intothe cytoplasmic male sterile parent or restored fertile parent bycrossing the parent plants with another corn plant (referred to as thedonor or non-recurrent parent) which carries the gene(s) encoding theparticular trait(s) of interest to produce F₁ progeny plants. Bothdominant and recessive alleles may be transferred by backcrossing. Thedonor plant may also be an inbred, but in the broadest sense can be amember of any plant variety or population cross-fertile with therecurrent parent. Next, F₁ progeny plants that have the desired traitare selected. Then, the selected progeny plants are crossed with thecytoplasmic male sterile parent or restored fertile parent to producebackcross progeny plants. Thereafter, backcross progeny plantscomprising the desired trait and the physiological and morphologicalcharacteristics of the cytoplasmic male sterile parent or restoredfertile parent are selected. This cycle is repeated for about one toabout eight cycles, preferably for about 3 or more times in successionto produce selected higher backcross progeny plants that comprise thedesired trait and all of the physiological and morphologicalcharacteristics of the cytoplasmic male sterile parent or restoredfertile parent when grown in the same environmental conditions.Exemplary desired trait(s) include insect resistance, enhancednutritional quality, waxy starch, herbicide resistance, yield stability,yield enhancement and resistance to bacterial, fungal and viral disease.One of ordinary skill in the art of plant breeding would appreciate thata breeder uses various methods to help determine which plants should beselected from the segregating populations and ultimately which inbredlines will be used to develop hybrids for commercialization. In additionto the knowledge of the germplasm and other skills the breeder uses, apart of the selection process is dependent on experimental designcoupled with the use of statistical analysis. Experimental design andstatistical analysis are used to help determine which plants, whichfamily of plants, and finally which inbred lines and hybrid combinationsare significantly better or different for one or more traits ofinterest. Experimental design methods are used to assess error so thatdifferences between two inbred lines or two hybrid lines can be moreaccurately determined. Statistical analysis includes the calculation ofmean values, determination of the statistical significance of thesources of variation, and the calculation of the appropriate variancecomponents. Either a five or a one percent significance level iscustomarily used to determine whether a difference that occurs for agiven trait is real or due to the environment or experimental error. Oneof ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr, Walt, Principles of CultivarDevelopment, p. 261-286 (1987) which is incorporated herein byreference. Mean trait values may be used to determine whether traitdifferences are significant, and preferably the traits are measured onplants grown under the same environmental conditions.

This method results in the generation of progeny inbred plants withsubstantially all of the desired morphological and physiologicalcharacteristics of the recurrent parent and the particular transferredtrait(s) of interest. Because such progeny inbred plants areheterozygous for loci controlling the transferred trait(s) of interest,the last backcross generation would subsequently be selfed to providepure breeding progeny for the transferred trait(s).

Backcrossing may be accelerated by the use of genetic markers such asSSR, RFLP, SNP or AFLP markers to identify plants with the greatestgenetic complement from the recurrent parent.

Direct selection may be applied where a single locus acts as a dominanttrait, such as the herbicide resistance trait. For this selectionprocess, the progeny of the initial cross are sprayed with the herbicidebefore the backcrossing. The spraying eliminates any plants which do nothave the desired herbicide resistance characteristic, and only thoseplants which have the herbicide resistance gene are used in thesubsequent backcross. In the instance where the characteristic beingtransferred is a recessive allele, it may be necessary to introduce atest of the progeny to determine if the desired characteristic has beensuccessfully transferred. The process of selection, whether direct orindirect, is then repeated for all additional backcross generations.

It should be appreciated by those having ordinary skill in the art thatbackcrossing can be combined with pedigree breeding as where thecytoplasmic male sterile parent or restored fertile parent is crossedwith another corn plant, the resultant progeny are crossed back to thecytoplasmic male sterile parent or restored fertile parent andthereafter, the resulting progeny of this single backcross aresubsequently inbred to develop new inbred lines. This combination ofbackcros sing and pedigree breeding is useful as when recovery of fewerthan all of the cytoplasmic male sterile parent or restored fertileparent characteristics than would be obtained by a conventionalbackcross are desired.

In an additional embodiment, the subject disclosure relates to a methodfor restoring fertility to a progeny of a cytoplasmic male sterileparent plant, the method comprising:

-   -   a) crossing a female parent plant with a male parent plant,        wherein the female parent plant is a cytoplasmic male sterile        parent plant homozygous or heterozygous for SEQ ID NO:8, and        wherein the cytoplasmic male sterile plant is a fertile parent        plant;    -   b) harvesting a progeny seed from the cross of (a), wherein the        progeny seed is homozygous or heterozygous for SEQ ID NO:8;    -   c) planting the progeny seed;    -   d) growing the progeny seed, wherein the progeny seed produce a        progeny cytoplasmic male fertile plant; and,    -   e) restoring fertility to the progeny of the cytoplasmic male        sterile parent plant, wherein the progeny cytoplasmic male        fertile plant is homozygous or heterozygous for SEQ ID NO:8.        Additional embodiments to the method include.    -   f) crossing the progeny cytoplasmic male sterile plant, with        another plant comprising a desired trait to produce F1 progeny        plants;    -   g) selecting F1 progeny plants that have the desired trait to        produce selected F1 progeny plants;    -   h) crossing the selected F1 progeny plants with progeny        cytoplasmic male sterile plant to produce backcross progeny        plants;    -   i) selecting for backcross progeny plants that have the desired        trait; and,    -   j) repeating steps (h) and (i) three or more times in succession        to produce selected fourth or higher backcross progeny plants        that comprise the desired trait.

In additional embodiments, the subject disclosure relates to female andmale parent plants that are monocotyledonous plants. In otherembodiments, the monocotyledonous plants are maize plants. In furtherembodiments the male parent plant is isogenic to the female parentplant. In an aspect of the embodiment, the male parent plant ishomozygous or heterozygous for SEQ ID NO:8. In a subsequent embodiment,the disclosure related to introducing a desired trait into the progenycytoplasmic male sterile plant. In an aspect of the embodiment, thedesired trait is selected from the group consisting of an insecticidalresistance trait, herbicide tolerant trait, disease resistance trait,yield increase trait, nutritional quality trait, agronomic increasetrait, and combinations thereof.

VII. Assays for Detection of the Rf3 and Rf3 Alleles

Various assays can be employed to detect the polynucleotides that encodethe Rf3 and rf3 alleles that are described in certain embodiments of thedisclosure. The following techniques are useful in a variety ofsituations, and in one embodiment, are useful in detecting the presenceof a nucleic acid molecule and/or the polypeptide encoding an Rf3 or rf3allele in a plant cell. For example, the presence of the molecule can bedetermined in a variety of ways, including using a primer or probe ofthe sequence, ELISA assay to detect the encoded protein, a Western blotto detect the protein, or a Northern or Southern blot to detect RNA orDNA. Enzymatic assays for detecting the polynucleotides that encode theRf3 and rf3 allele can be employed. Further, an antibody which candetect the presence of the Rf3 or rf3 protein can be generated using artrecognized procedures. Additional techniques, such as in situhybridization, enzyme staining, and immunostaining, also may be used todetect the presence or expression of the polynucleotides that encode theRf3 and rf3 alleles in specific plant organs and tissues. Thepolynucleotides that encode the Rf3 and rf3 alleles may be selectivelyexpressed in some tissues of the plant or at some developmental stages,or the polynucleotides that encode the Rf3 and rf3 alleles may beexpressed in substantially all plant tissues, substantially along itsentire life cycle. However, any combinatorial expression mode is alsoapplicable.

In an embodiment the disclosure relates method of detecting a plantcomprising a cytoplasmic male sterile restorer trait via anamplification reaction in which an amplified product or amplicon isgenerated. The detection of the absence of the amplicon is an indicationof whether the plant contains an Rf3 or rf3 allele of gene sequence,respectively.

Various assays can be employed in connection with the amplificationreaction and are considered as embodiments of the disclosure. Thefollowing techniques are useful in a variety of situations, and in oneembodiment, are useful in detecting the presence of the nucleic acidmolecule and/or the polypeptide encoded in a plant cell. For example,the presence of the molecule can be determined in a variety of ways,including using a primer or probe of the sequence. The Rf3 or rf3alleles may be selectively expressed in some tissues of the plant or atsome developmental stages, or the Rf3 or rf3 alleles may be expressed insubstantially all plant tissues, throughout the entire life cycle of theplant. However, any combinatorial expression mode is also applicable.

Amplification of a nucleic acid sequence may be carried out by anysuitable means. See generally, Kwoh et al., Am. Biotechnol. Lab. 8,14-25 (1990). Examples of suitable amplification techniques include, butare not limited to, polymerase chain reaction, ligase chain reaction,strand displacement amplification (see generally G. Walker et al., Proc.Natl. Acad. Sci. USA 89, 392-396 (1992); G. Walker et al., Nucleic AcidsRes. 20, 1691-1696 (1992)), transcription-based amplification (see D.Kwoh et al., Proc. Natl. Acad Sci. USA 86, 1173-1177 (1989)),self-sustained sequence replication (or “35R”) (see J. Guatelli et al.,Proc. Natl. Acad. Sci. USA 87, 1874-1878 (1990)), the Qβ replicasesystem (see P. Lizardi et al., BioTechnology 6, 1197-1202 (1988)),nucleic acid sequence-based amplification (or “NASBA”) (see R. Lewis,Genetic Engineering News 12 (9), 1 (1992)), the repair chain reaction(or “RCR”) (see R. Lewis, supra), and boomerang DNA amplification (or“BDA”) (see R. Lewis, supra). Polymerase chain reaction is generallypreferred.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques were designed primarily for this sorting out.

As used herein, the term “polymerase chain reaction” and “PCR” generallyrefers to the method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification (U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188; hereinincorporated by reference). This process for amplifying the targetsequence comprises introducing an excess of two oligonucleotide primersto the DNA mixture containing the desired target sequence, followed by aprecise sequence of thermal cycling in the presence of a DNA polymerase.The two primers are complementary to their respective strands of thedouble stranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction” (hereinafter “PCR”).Because the desired amplified segments of the target sequence become thepredominant sequences (in terms of concentration) in the mixture, theyare said to be “PCR amplified.

The term “plurality” is used herein to mean two or more, for example,three, four, five or more, including ten, twenty, fifty or morepolynucleotides, nucleic acid probes, and the like.

The term “reverse-transcriptase” or “RT-PCR” refers to a type of PCRwhere the starting material is mRNA. The starting mRNA is enzymaticallyconverted to complementary DNA or “cDNA” using a reverse transcriptaseenzyme. The cDNA is then used as a “template” for a “PCR” reaction.

In an embodiment, the amplification reaction is quantified. In otherembodiments, the amplification reaction is quantitated using a signatureprofile, in which the signature profile is selected from the groupconsisting of a melting temperature or a fluorescence signature profile.In further embodiments the amplification reaction is quantitated theDelta Delta ct method.

The polynucleotides that encode the Rf3 and rf3 genes, or segmentsthereof, can be used as primers for PCR amplification. In performing PCRamplification, a certain degree of mismatch can be tolerated betweenprimer and template. Therefore, mutations, deletions, and insertions(especially additions of nucleotides to the 5′ end) of the exemplifiedprimers fall within the scope of the subject disclosure. Mutations,insertions, and deletions can be produced in a given primer by methodsknown to an ordinarily skilled artisan.

Another example of method detection is the pyrosequencing technique asdescribed by Winge (Innov. Pharma. Tech. 00:18-24, 2000). In this methodan oligonucleotide is designed that overlaps the adjacent genomic DNAand insert DNA junction. The oligonucleotide is hybridized tosingle-stranded PCR product from the region of interest and incubated inthe presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase,adenosine 5′ phosphosulfate and luciferin. DNTPs are added individuallyand the incorporation results in a light signal that is measured. Alight signal indicates the presence of the transgene insert/flankingsequence due to successful amplification, hybridization, and single ormulti-base extension (this technique is used for initial sequencing, notfor detection of a specific gene when it is known).

Molecular Beacons have been described for use in sequence detection.Briefly, a FRET oligonucleotide probe is designed that overlaps theflanking genomic and insert DNA junction. The unique structure of theFRET probe results in it containing a secondary structure that keeps thefluorescent and quenching moieties in close proximity. The FRET probeand PCR primers are cycled in the presence of a thermostable polymeraseand dNTPs. Following successful PCR amplification, hybridization of theFRET probe(s) to the target sequence results in the removal of the probesecondary structure and spatial separation of the fluorescent andquenching moieties. A fluorescent signal indicates the presence of theflanking genomic/transgene insert sequence due to successfulamplification and hybridization.

Hydrolysis probe assay, otherwise known as Taqman® (Life Technologies,Foster City, Calif.), is a method of detecting and quantifying thepresence of a DNA sequence. Briefly, a FRET oligonucleotide probe isdesigned with one oligo within the transgene and one in the flankinggenomic sequence for event-specific detection. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankinggenomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Hybridization of the FRET probe results incleavage and release of the fluorescent moiety away from the quenchingmoiety on the FRET probe. A fluorescent signal indicates the presence ofthe flanking/transgene insert sequence due to successful amplificationand hybridization.

The ELISA or enzyme linked immunoassay has been known since 1971. Ingeneral, antigens solubilised in a buffer are coated on a plasticsurface. When serum is added, antibodies can attach to the antigen onthe solid phase. The presence or absence of these antibodies can bedemonstrated when conjugated to an enzyme. Adding the appropriatesubstrate will detect the amount of bound conjugate which can bequantified. A common ELISA assay is one which uses biotinylatedanti-(protein) polyclonal antibodies and an alkaline phosphataseconjugate. For example, an ELISA used for quantitative determination oflaccase levels can be an antibody sandwich assay, which utilizespolyclonal rabbit antibodies obtained commercially. The antibody isconjugated to alkaline phosphatases for detection. In another example,an ELISA assay to detect trypsin or trypsinogen uses biotinylatedanti-trypsin or anti-trypsinogen polyclonal antibodies and astreptavidin-alkaline phosphatase conjugate.

KASPar® assays are a method of detecting and quantifying the presence ofa DNA sequence. Briefly, the genomic DNA sample comprising the targetedgenomic locus is screened using a polymerase chain reaction (PCR) basedassay known as a KASPar® assay system. The KASPar® assay used in thepractice of the subject disclosure can utilize a KASPar® PCR assaymixture which contains multiple primers. The primers used in the PCRassay mixture can comprise at least one forward primers and at least onereverse primer. The forward primer contains a sequence corresponding toa specific region of the DNA polynucleotide sequence, and the reverseprimer contains a sequence corresponding to a specific region of asecond DNA polynucleotide sequence. In addition, the primers used in thePCR assay mixture can comprise at least one forward primers and at leastone reverse primer. For example, the KASPar® PCR assay mixture can usetwo forward primers corresponding to two different alleles and onereverse primer. One of the forward primers contains a sequencecorresponding to specific region of the endogenous genomic sequence. Thesecond forward primer contains a sequence corresponding to a specificregion of the donor DNA polynucleotide. The reverse primer contains asequence corresponding to a specific region of the genomic sequence.Such a KASPar® assay for detection of an amplification reaction is anembodiment of the subject disclosure.

In some embodiments the fluorescent signal or fluorescent dye isselected from the group consisting of a HEX fluorescent dye, a FAMfluorescent dye, a JOE fluorescent dye, a TET fluorescent dye, a Cy 3fluorescent dye, a Cy 3.5 fluorescent dye, a Cy 5 fluorescent dye, a Cy5.5 fluorescent dye, a Cy 7 fluorescent dye, and a ROX fluorescent dye.

In other embodiments the quencher is selected from the group consistingof a Dabcyl quencher, a Tamra quencher, a Qxl quencher, an Iowa Black FQquencher, an Iowa Black RQ quencher, an IR Dye QC-1 quencher, a MGBquencher, or a Blackhole quencher.

In other embodiments the amplification reaction is run using suitablesecond fluorescent DNA dyes that are capable of staining cellular DNA ata concentration range detectable by flow cytometry, and have afluorescent emission spectrum which is detectable by a real timethermocycler. It should be appreciated by those of ordinary skill in theart that other nucleic acid dyes are known and are continually beingidentified. Any suitable nucleic acid dye with appropriate excitationand emission spectra can be employed, such as YO-PRO-1®, SYTOX Green®,SYBR Green I®, SYTO11®, SYTO12®, SYTO13®, BOBO®, YOYO®, and TOTO®. inone embodiment, a second fluorescent DNA dye is SYTO13® used at lessthan 10 μM, less than 4 μM, or less than 2.7 μM.

In various embodiments, other known detection methods are performed todetect the Rf3 or rf3 alleles.

Southern analysis is a commonly used detection method, wherein DNA iscut with restriction endonucleases and fractionated on an agarose gel toseparate the DNA by molecular weight and then transferring to nylonmembranes. It is then hybridized with the probe fragment which wasradioactively labeled with 32P (or other probe labels) and washed in anSDS solution.

Likewise, Northern analysis deploys a similar protocol, wherein RNA iscut with restriction endonucleases and fractionated on an agarose gel toseparate the RNA by molecular weight and then transferring to nylonmembranes. It is then hybridized with the probe fragment which wasradioactively labeled with 32P (or other probe labels) and washed in anSDS solution. Analysis of the RNA (e.g., mRNA) isolated from the tissuesof interest can indicate relative expression levels. Typically, if themRNA is present or the amount of mRNA has increased, it can be assumedthat the corresponding transgene is being expressed. Northern analysis,or other mRNA analytical protocols, can be used to determine expressionlevels of an introduced transgene or native gene.

In the Western analysis, instead of isolating DNA/RNA, the protein ofinterest is extracted and placed on an acrylamide gel. The protein isthen blotted onto a membrane and contacted with a labeling substance.See e.g., Hood et al., “Commercial Production of Avidin from TransgenicMaize; Characterization of Transformants, Production, Processing,Extraction and Purification” Molecular Breeding 3:291-306 (1997); Towbinet al, (1979) “Electrophoretic transfer of proteins from polyacrylamidegels to nitrocellulose sheets: procedure and some applications” ProcNatl Acad Sci USA 76(9): 4350-4354; Renart et al. “Transfer of proteinsfrom gels to diazobenzyloxymethyl-paper and detection with antisera: amethod for studying antibody specificity and antigen structure” ProcNatl Acad Sci USA 76(7): 3116-3120.

Embodiments of the subject disclosure are further exemplified in thefollowing Examples. It should be understood that these Examples aregiven by way of illustration only. From the above embodiments and thefollowing Examples, one skilled in the art can ascertain the essentialcharacteristics of this disclosure, and without departing from thespirit and scope thereof, can make various changes and modifications ofthe embodiments of the disclosure to adapt it to various usages andconditions. Thus, various modifications of the embodiments of thedisclosure, in addition to those shown and described herein, will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims. The following is provided by way of illustration andnot intended to limit the scope of the invention.

EXAMPLES Example 1: Development of a Backcross 1 Population for Mappingand Fine Resolution of the Rf3 Alleles

A backcross 1 (BC1) population was produced and utilized via linkage mapanalysis with developed molecular markers to map the Rf3 codingsequence. The mapping population was created by crossing a maizecytoplasmic male sterile line, Zea mays c.v. 4XP811 (U.S. Pat. No.7,135,629; that contained homozygous recessive rf3 alleles) with a maizerestoring line Zea mays c.v. MBB56 (that contained at least one dominantRf3 allele), and then backcrossing progeny with the maize cytoplasmicmale sterile line, Zea mays c.v. 4XP811. The resultant population ofmaize progeny contained 275 individuals with varying degrees of zygosityfor the Rf3 (dominant) and rf3 (recessive) alleles.

Resulting maize plants were phenotypically classified according to twomethods. The first method involved observation of the shedding pollenfrom the maize plants in the field. Fertile plants were observed for thegrowth and development of anthers that were physically exposed beforesilk development and the start of pollen shedding. Cytoplasmic malesterile plants were observed for delayed growth and development ofanthers. By the middle of silk development (e.g., around 7 days afterthe silks begin to develop) the anthers were not physically exposed.

The second method involved determination of the vitality of pollen grainby using 1% KI-I₂ stain. When the pollen of fertile plants was stainedwith a KI-I₂ stain, the pollen absorbed the KI-I₂ stain therebyresulting in uniformly stained pollen. It is well known that the pollenfrom fertile plants is filled with starch to which the KI-I₂ stainadheres. When the pollen from cytoplasmic male sterile plants wasstained, the pollen did not absorb the KI-I₂ stain thereby resulting inunstained pollen. The pollen of the cytoplasmic male sterile plantscontain collapsed pollen that contains low levels of starch, and theKI-I₂ stain cannot bind to the minimal amounts of starch present in thepollen. Table 1 provides the detailed segregation information from theBC1 mapping population.

TABLE 1 Phenotype data of the BC1 mapping population. Sterile(homozygous Fertile recessive, rf3/rf3) (heterozygous) cytoplasmic maleBroken BC1 Population restorer plants sterile plants plants 4XP811 ×BMM56 139 120 16

The resulting data indicated that fertility segregation of the BC1population fit a theoretical 1:1 ratio of fertile plants:sterile plantsusing the one-gene segregation model (X²=1.39 [a (0.05)=3.84]). The datafurther suggested that the germplasm of the MBB56 line carried onerestorer gene, Rf3, and the germplasm of the 4XP811 was homozygous forthe rf3 gene.

Example 2: Marker Assisted Genome Mapping of RF3

The BC1 populations were screened with molecular markers to identify thelocus corresponding with the RF3 allele to high resolution. A panel of482 SNP markers (evenly distributed on the whole genome) was selected togenotype the BC1 mapping population. From this assay, 437 SNP markers(including four PCR-based markers from PPR2, 432 SNP based markers fromacross the genome, and one marker based on sterile-fertile phenotype ofthe BC1 mapping population) were used to create a genetic linkage withJoinMap 4.0 Software™ (Van Ooijen J W: JoinMap 4. Software for thecalculation of genetic linkage maps in experimental populations KyazmaBV. Netherlands: Wageningen; 2006). Utilizing the genetic linkage map,the phenotypic data, and the genotypic data, a whole genome QTL analysiswas performed using Map QTL 6.0™ software (Van Ooijen J W: Map QTL 6.Wageningen, Netherlands: Software for the mapping of quantitative traitloci in experimental populations of diploid species Kyazma BV; 2009).The results showed that all the PPR2 gene specific markers co-segregatedwith the RF3 locus. The genomic location of RF3 locus was determined tolie on chromosome 2 in a 1.3 Mb genomic interval of the corn genome (seeFIG. 1B).

Example 3: Whole Genome Sequencing to Identify the RF3 Gene Sequence

To further identify the specific Rf3 restorer gene sequence and the genesequence of the rf3 modification which results in cytoplasmic malesterility, Next Generation Sequencing (NGS) technology was utilized tofurther investigate the Rf3 locus. The genomic sequences of two maizeCMS-S lines and two maize restorer lines were determined using NGSsequencing. The two CMS-S lines (4XP811 and 7SH382 ms) and the tworestorer lines (LH60 and MBB56) were used for whole genome sequencinganalysis.

A total of 10 PPR genes (annotated sequentially as PPR 1-10) wereidentified on chromosome 2 within a 1.3 Mb genomic interval aftercomparison of the sequenced genomic regions with annotated sequence ofthe reference genome from Zea mays c.v. B73 (available atwww.maizegdb.org). A full length coding sequence that corresponds withPPR2 did not exist in the annotated Zea mays c.v. B73 genome and couldnot be predicted as a full length, functional gene. As a result, thefull gene sequence for PPR2 was obtained from a sequence comparison tothe reference genome of Zea mays c.v. Mo17 (available atwww.maizegdb.org). The resulting sequences for the 10 PPR genes (PPR2had two sequences, the first sequence from line Zea mays c.v. B73 andthe second sequence from line Zea mays c.v. Mo17) were assembled forreference sequence information based on whole genome sequencing data ofthe two CMS-S lines and two restorer lines.

Next, the sequences were aligned between CMS-S lines, restorer lines,and the reference genomic sequences for all of the PPR genes. Codingsequence prediction (e.g., cDNA wherein the intron sequences wereremoved) of the PPR genomic sequences was completed using Softberry™software (Softberry Inc., Mount Kisco, N.Y.), and the predicted cDNA andcorresponding protein sequences were obtained and aligned. Genomicsequence variations were identified from the aligned genomic sequencesof the PPR genes and noted. When the cDNA and the protein sequences werealigned between CMS-S and restorer lines with reference sequences, itwas observed that the majority of the variations within the PPR2sequences resulted in silent mutations (see FIG. 2 and FIG. 3).Variations in the PPR2 sequences that resulted in amino acid residuemodifications were identified, and RT-PCR assays were developed todetect and amplify these modifications (e.g., mutations).

The results of the genetic linkage mapping and NGS indicated that theRf3 allele and the subsequent CMS phenotype was the result of a singlegene located on chromosome 2 Like the majority of the cloned Rf genes,Rf3 is most likely a PPR gene. The restorer Rf3-PPR gene from Zea maysc.v. LH60 (SEQ ID NO:8) was identified to be present in restorer,fertile plant phenotypes and encodes an 814-amino acid protein (SEQ IDNO: 7). In addition, a partial polynucleotide fragment of the restorerRf3-PPR gene from Zea mays c.v. MBB56 (SEQ ID NO:12) was identified tobe present in restorer, fertile plant phenotypes and encodes the aminoacid protein fragment (SEQ ID NO: 11). An amino acid motif, SEQ ID NO:5(IVLFSS), which is encoded by SEQ ID NO:6 (5′-ATTGTTTTATTCAGT-3′), wasfound to be common for both of the wild type Rf3-PPR genes from Zea maysc.v. LH60 and Zea mays c.v. MBB56.

The mutated, cytoplasmic male sterile rf3-PPR gene from Zea mays c.v.4XP811 (SEQ ID NO:4) was identified to be present in cytoplasmic malesterile plant phenotypes and encodes an 814-amino acid protein (SEQ IDNO:3). In addition, a cytoplasmic male sterile rf3-PPR gene from Zeamays c.v. 7SH382 ms (SEQ ID NO:10) was identified to be present incytoplasmic male sterile plant phenotypes and encodes an 814-amino acidprotein (SEQ ID NO:9). An amino acid motif, SEQ ID NO:1 (IVFFSS), whichis encoded by SEQ ID NO:2 (5′-ATTGTTTTCTTCAGT-3′), was found to becommon for both of the Rf3-PPR genes from Zea mays c.v. 4XP811 and Zeamays c.v. 7SH382 ms.

Example 4: Expression Analysis of RF3-PPR2 by Real-Time PCR

To validate whether the expression of the Rf3-PPR2 protein correlatedwith fertility restoration of S-CMS maize, a real-time PCR (RT-PCR)assay was performed to quantitatively determine the expression patternof the Rf3-PPR2 gene in fertile plants and the rf3-PPR2 gene in CMSplants. Several specific primer pairs and probes were designed based onportions of the polynucleotide sequences that contained amino acids withvariations in the Rf3-PPR2 protein coding gene (Table 2). Total RNA wasextracted from the various plant lines. These samples were extractedfrom two lines; 4XP811 (cytoplasmic male sterile) and LH60 (restorerlines), F3 individuals derived from an F2 ear segregating for the 1.3-Mbregion of long arm chromosome 2, and three commercial maize lines.Taqman® assays to quantitate the expression of the Rf3-PPR2 gene werecompleted. The expression levels were quantitated by comparison to amaize internal control gene, EF α1. (Czechowski T, et al., PlantPhysiol., September; 139(1):5-17, 2005).

TABLE 2 Primers and probes used for RT-PCR of the RF3 maize plants.Primer SEQ Reaction Name Primer Sequence ID NO: CMS-S1 ForwardGATTGATCAAGGAG 13 Primer TAGCACCTGA Reverse CTTGGCTTTCAGTA 14 PrimerAACTACCATGAGT Probe CATACCATTGCCTG 15 ATTC CMS-S2 Forward GGTAGTTTACTGAA16 Primer AGCCAAGGAATT Reverse TTGCAAAGGTTGTT 17 Primer AATTATCGAACTProbe ATGGCATGCATCTT 18 GACAT CMS-S3 Forward GGGTACTGTCTTGT 19 PrimerTGGCAAGAT Reverse TGGTTCAATGCCAG 20 Primer CTGACA Probe AGAATGCATTAAGA21 GTATTTGATGC CMS-S7 Forward GGACAGTGGAAGGA 22 Primer GGCAGTTA ReverseCCATACTTGCAAAG 23 Primer GGAACCC Probe CCAGATGTTGTTAC 24 TTTTAACATGCMS-S8 Forward GAATAATGGCATGC 25 Primer GTCTTGATATT ReverseGTAGGATGCAGACC 26 Primer AACATTTACAGT Probe CCTTTGCAAATTGG 27 GAAGRF3 allele Forward GTACTCATGGTAGT 28 specific: Primer TTACTGAAAGCCACMS-S9 Reverse GCATCCATTACCCT 29 Primer TCCCAAT Probe ATCTTGACATTGTT 30TTATTCAGTTCG CMS-S10 Forward GTTCCTGCAAAGGT 31 Primer GAAATTCC ReverseGAAAGATTGCTTCA 32 Primer TCAAAGCATC Probe GGTATCGCTATGTA 33 CATATGInternal Forward ATAACGTGCCTTGG 34 Control: Primer AGTATTTGG ElongationReverse TGGAGTGAAGCAGA 35 Factor α-1 Primer TGATTTGC ProbeTTGCATCCATCTTG 36 TTGC

The plants to be analyzed via the Taqman® assay were grown in agreenhouse. Leaf tissues were collected from 7-week old (just beforetasselling) and 10-week old plants (after pollination). Tassel tissueswith developing anthers/pollens and shed pollens (in fertile plants)were also collected. Total RNA was extracted using Qiagen RNeasy PlantMini Kit™ and cDNA was synthesized using Qiagen QuantiTect ReverseTranscription Kit™ (Qiagen, Carlsbad, Calif.). For RT-PCR, theexpression of elongation factor α-1 (EF α1) of maize was used as aninternal control. Primers for Rf3-PPR2 and EF α1 and dual labeled probeswith FAM or VIC dyes and Minor Groove Binding Non Fluorescence Quencher™I (MGBNFQ) quencher were synthesized by Applied Biosystems (Foster City,Calif.). Taqman® genotyping master mix (Applied Biosystems, Foster City,Calif.) was used to set up 10 μl PCR reactions and the PCR was performedon Roche LightCycler 480™ thermocycler (Roche, Indianapolis, Ind.). ThePCR program was initiated with 10 minutes activation of the Taq enzymeat 95° C., followed by 50 cycles of 95° C. for 10 seconds and 58° C. for38 seconds. Fluorescence signals were recorded at the end of each cycle.Relative expression levels of Rf3-PPR2 to EF α1 was calculated using theDelta Delta CT method.

Seven Taqman® assays were designed based on modifications that resultedin amino acid changes within the Rf3-PPR2 gene (see Table 2).Surprisingly, only the CMS-S9 assay amplified an amplicon thatcorresponded with the presence of Rf3 or rf3 gene sequence in thefertile restorer lines and cytoplasmic male sterile lines, respectively.This assay was able to identify a single base pair modification (e.g.,mutation) that resulted in the rf3 cytoplasmic male sterile phenotype.As such, this single base pair modification could be used to discernbetween the rf3 cytoplasmic male sterile and the Rf3 restored fertileplants. Despite the numerous amino acid mutation present in the rf3lines, only plants containing the SEQ ID NO:1 motif resulted in theobservation of rf3 cytoplasmic male sterile plants.

The results of the quantitative RT-PCR showed that plants containing theSEQ ID NO:5 motif expressed in Rf3 the restorer parent LH60 and F3plants that were homozygous or heterozygous for the restorer allele inall tested growing stages. However, Rf3 did not express in CMS-Sparents, F3s homozygous for the rf3 allele and the commercial maizelines. Notably, Rf3 expression levels were distinguishable between Rf3homozygous (Rf3/Rf3) and heterozygous (Rf3/rf3) F3 individuals, whichcould explain why homozygous (Rf3/Rf3) plants shed 100% starched-filledfertile pollen while heterozygous (Rf3/rf3) plants shed approximately50% starch-filled fertile pollen. Finally, the Rf3-PPR2 gene, whichrestores S-type CMS cytoplasm, expressed in both tassel tissuecontaining immature pollen, and leaf tissue.

While aspects of this invention have been described in certainembodiments, they can be further modified within the spirit and scope ofthis disclosure. This application is therefore intended to cover anyvariations, uses, or adaptations of embodiments of the invention usingits general principles. Further, this application is intended to coversuch departures from the present disclosure as come within known orcustomary practice in the art to which these embodiments pertains andwhich fall within the limits of the appended claims.

What is claimed is:
 1. A fusion polypeptide comprising an amino acidsequence having at least 97.5% sequence to the amino acid sequence ofSEQ ID NO: 3, wherein said fusion polypeptide comprises an amino acidmotif comprising the amino acid sequence of SEQ ID NO: 1; and whereinsaid fusion polypeptide comprises cytoplasmic male sterile activity. 2.The fusion polypeptide of claim 1, wherein said fusion polypeptide iscomprised by a cell.
 3. The fusion polypeptide of claim 1, wherein saidfusion polypeptide is encoded by a synthetic nucleic acid sequence. 4.The fusion polypeptide of claim 3, wherein said synthetic nucleic acidsequence is operably linked to a promoter.
 5. The fusion polypeptide ofclaim 4, wherein said promoter is functional in plants.
 6. The fusionpolypeptide of claim 5, wherein said promoter is a cytoplasmic malesterile gene promoter.